Systems and methods for quality control in single cell processing

ABSTRACT

Provided are systems and methods for analyzing a single cell application or experiment. A set of control beads may be introduced to a biological sample and subjected to the single cell application. The control beads may be configured to mimic analytes in the biological sample, such as a cell or other analyte, and comprise one or more known sequences. The one or more known sequences may be identified to analyze the single cell application.

CROSS-REFERENCE

This application is a continuation of international patent applicationno. PCT/US2019/025180, filed Apr. 1, 2019, which claims the benefit ofU.S. Provisional Application No. 62/653,815, filed Apr. 6, 2018, each ofwhich is entirely incorporated herein by reference for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Jul. 29, 2019, is named43487794301SL.txt and is 2,599 bytes in size.

BACKGROUND

A sample may be processed for various purposes, such as identificationof a type of moiety within the sample. The sample may be a biologicalsample. Biological samples may be processed, such as for detection of adisease (e.g., cancer) or identification of a particular species. Thereare various approaches for processing samples, such as polymerase chainreaction (PCR) and sequencing. The processing may be single cellprocessing, such as to yield data specific and distinct to individualcells.

Biological samples may be processed within various reactionenvironments, such as partitions. Partitions may be wells or droplets.Droplets or wells may be employed to process biological samples in amanner that enables the biological samples to be partitioned andprocessed separately. For example, such droplets may be fluidicallyisolated from other droplets, enabling accurate control of respectiveenvironments in the droplets.

Biological samples in partitions may be subjected to various processes,such as chemical processes or physical processes. Samples in partitionsmay be subjected to heating or cooling, or chemical reactions, such asto yield species that may be qualitatively or quantitatively processed.

SUMMARY

Single cell processing experiments are becoming increasingly common andefficient. Massively parallel sequencing or next-generation sequencing,for example, allow for high-throughput processing that can yieldthousands, tens of thousands, hundreds of thousands, or millions of, ormore base reads in a single run. However, as is often the case, theresults (e.g., data) from an experiment are only as reliable as theexperimental process itself can be validated, that is, that theexperiment performed as designed. For example, experiments may havedifferent sensitivities, accuracies, and/or biases, which are notnecessarily, if at all, reflected in the data results. Given therelatively miniscule scale and high throughput of a single cell assay,as well as the irreproducible nature of some of these assays which canconsume or contaminate unique samples during an immediate assay, it isdifficult to validate or investigate, or otherwise perform qualitycontrol (QC) on, how well an experiment performs during the single cellassay or be able to compare two or more different single cell assays, orresults thereof.

One approach for quality control of a single cell sequencing experimentmay involve introducing a control into the sample to be sequenced, andtracking the control. For example, the External RNA Controls Consortium(ERCC) has developed ribonucleic acid (RNA) control transcripts (ERCCRNA Spike-In Controls) with known sequences that may be spiked into RNAsamples for input into a RNA sequencing (RNA-seq) experiment. Theresults of a spiked-in RNA-seq experiment may then be compared to theknown control sequences, such as to determine whether the experiment wasable to properly detect the control, or at what dosages of controls theexperiment was able to detect the control to assess sensitivity, and thelike. Another approach for quality control may involve introducing anexogenous genome or epigenome as reference into the sample, such as fornormalizing chromatin immunoprecipitation sequencing (ChIP-seq)analyses. However, such methods of control fail to mimic true singlecell behavior—it would be more accurate to describe the above methods asperforming quality control on the treatment of an analyte in a cell(e.g., nucleic acids, chromatins, etc.), and not the cell, in the singlecell experiment. For example, the known nucleic acids (e.g., knowntranscripts) are distributed uniformly in equal amounts to allpartitions, including partitions that already contain cells, inherentlybiasing the single cell experiment. Furthermore, using bulk ERCCcontrols require carefully controlling the input concentration andsubsampling to manage sequencing requirements, both of which introducehandling variables that can affect quality control. Additionally, usingbulk ERCC controls does not allow for quality control of themicrofluidics involved in the single cell experiment.

Recognized herein is a need for systems and methods for quality controlof single cell processing experiments that addresses at least theabovementioned problems. Provided are synthetic cells that mimic singlecell behavior in single cell processing experiments. A synthetic cellmay be a bead comprising one or more known sequences. The bead may be agel bead. The synthetic cell may be introduced into a cell sample andhave approximately the same or substantially the same size as othercells in the cell sample. The synthetic cell may be carried through theentire workflow of a single cell processing experiment with the cellsample. After sequencing, the one or more known sequences in thesynthetic cell may be identified and analyzed to determine variouscharacteristics of the single cell processing experiment, such as theeffectiveness or the efficiency of the library preparation process orthe sequencing process.

In an aspect, provided is a method for analyzing a single cell processin a cell sample, comprising: (a) providing a plurality of analytecarriers, a plurality of control beads, and a plurality of barcodebeads; (b) generating a plurality of partitions comprising a firstpartition and a second partition, wherein (i) the first partitioncomprises an analyte carrier from the plurality of analyte carriers anda first barcode bead from the plurality of barcode beads, wherein theanalyte carrier comprises a template nucleic acid molecule, and whereinthe first barcode bead comprises a first nucleic acid barcode moleculecomprising a first barcode sequence, and (ii) the second partitioncomprises a control bead from the plurality of control beads and asecond barcode bead from the plurality of barcode beads, wherein thesecond barcode bead comprises a second nucleic acid barcode moleculecomprising a second barcode sequence, wherein the second barcodesequence is different than the first barcode sequence, and wherein thecontrol bead comprises a nucleic acid molecule comprising a knownsequence; (c) using (i) the template nucleic acid molecule and the firstnucleic acid barcode molecule to generate a barcoded template nucleicacid molecule and (ii) the nucleic acid molecule comprising the knownsequence and the second nucleic acid barcode molecule to generate abarcoded nucleic acid molecule comprising the known sequence; and (d)sequencing the barcoded template nucleic acid molecule, or derivativethereof, and the barcoded nucleic acid molecule, or derivative thereofwherein (i) a sequence of the barcoded template nucleic acid moleculeidentifies the analyte carrier, and (ii) a sequence of the barcodednucleic acid molecule identifies the control bead.

In some embodiments, the method further comprises identifying the knownsequence to analyze the single cell process.

In some embodiments, the method further comprises providing a pluralityof second control beads, wherein the plurality of partitions comprises athird partition, wherein the third partition comprises a third barcodebead from the plurality of barcode beads and a second control bead fromthe plurality of second control beads, wherein the third barcode beadcomprises a third nucleic acid barcode molecule comprising a thirdbarcode sequence, wherein the third barcode sequence is different thanthe first barcode sequence and the second barcode sequence, and whereinthe second control bead comprises a second nucleic acid moleculecomprising a second known sequence, wherein the second known sequence isdifferent from the known sequence.

In some embodiments, the method further comprises generating a secondbarcoded nucleic acid molecule comprising the second known sequence,wherein a sequence of the second barcoded nucleic acid moleculeidentifies the second known sequence. In some embodiments, the methodfurther comprises processing the known sequence and the second knownsequence to analyze the single cell process. In some embodiments, themethod further comprises processing comprises comparing a frequency ofthe known sequence and a frequency of the second known sequence toanalyze the single cell process. In some embodiments, the processingfurther comprises determining a doublet rate.

In some embodiments, the second control bead has a size within about 70%deviation from an average size of the analyte carrier. In someembodiments, the second control bead has a size within about 40%deviation from an average size of the analyte carrier. In someembodiments, the second control bead has a size within about 25%deviation from the average size of the analyte carrier. In someembodiments, the second control bead has a size within about 10%deviation from the average size of the analyte carrier. In someembodiments, the second control bead has a size within about 5%deviation from the average size of the analyte carrier.

In some embodiments, the size of the second control bead has a sizewithin about 40% deviation from an average size of the control bead. Insome embodiments, the second control bead has a size within about 25%deviation from the average size of the control bead. In someembodiments, the second control bead has a size within about 10%deviation from the average size of the control bead. In someembodiments, the second control bead has a size within about 5%deviation from the average size of the control bead.

In some embodiments, the known sequence is derived from a first speciesand the second known nucleic acid sequence is derived from a secondspecies. In some embodiments, the first species is a human and whereinthe second species is a mouse.

In some embodiments, the control bead has a size within about 40%deviation from an average size of the analyte carrier. In someembodiments, the control bead has a size within about 25% deviation fromthe average size of the analyte carrier. In some embodiments, thecontrol bead has a size within about 10% deviation from the average sizeof the analyte carrier. In some embodiments, the control bead has a sizewithin about 5% deviation from the average size of the analyte carrier.

In some embodiments, the control bead has a size between from about 15micrometers to about 35 micrometers. In some embodiments, the controlbead has a size between from about 35 micrometers to about 60micrometers.

In some embodiments, a given bead from the plurality of barcode beadscomprises a plurality of nucleic acid barcode molecules comprising acommon barcode sequence. In some embodiments, the common barcodesequence is different from common barcode sequences of other beads ofthe plurality of beads.

In some embodiments, the method further comprises, prior to (b), mixinganalyte carriers from the plurality of analyte carriers with controlbeads of the plurality of control beads.

In some embodiments, the method further comprises, prior to (b), mixinganalyte carriers from the plurality of analyte carriers with controlbeads of the plurality of control beads and control beads of the secondplurality of control beads. In some embodiments, the control beads fromthe plurality of control beads is present in the mixture at a firstconcentration, wherein the control beads from the second plurality ofcontrol beads is present in the mixture at a second concentration, andwherein the first concentration and the second concentration are known.In some embodiments, a ratio of the first concentration to the secondconcentration is at least about 1:0.001. In some embodiments, a ratio ofa concentration of the analyte carriers in the mixture to the firstconcentration is known. In some embodiments, the ratio is about 0.001:1.

In some embodiments, the control bead comprises a plurality of uniquenucleic acid molecules with known nucleic acid sequences that vary inlength, concentration, and/or GC content. In some embodiments, theplurality of unique nucleic acid molecules is a plurality of DNAmolecules. In some embodiments, the plurality of unique nucleic acidmolecules is a plurality of RNA molecules.

In some embodiments, (i) the control bead comprises a first plurality ofunique nucleic acid molecules with known nucleic acid sequences thatvary in length, concentration, and/or GC content, and wherein (ii) thesecond control bead comprises a second plurality of unique nucleic acidmolecules with known nucleic acid sequences that vary in length,concentration, and/or GC content, wherein the first plurality of uniquenucleic acid molecules and the second plurality of unique nucleic acidmolecules are different.

In some embodiments, the first plurality of unique nucleic acidmolecules and the second plurality of unique nucleic acid molecules areboth a plurality of DNA molecules. In some embodiments, the firstplurality of unique nucleic acid molecules and the second plurality ofunique nucleic acid molecules are both a plurality of RNA molecules.

In some embodiments, a given bead from the plurality of barcode beadscomprises a plurality of nucleic acid barcode molecules, wherein each ofthe plurality of nucleic acid barcode molecules comprises an identifiersequence, wherein the identifier sequence is different from identifiersequences associated with other nucleic acid barcode molecules in theplurality of nucleic acid barcode molecules. In some embodiments, themethod further comprises identifying a first set of identifiersassociated with the first plurality of unique nucleic acid molecules anda second set of identifiers associated with the second plurality ofunique nucleic acid molecules. In some embodiments, the method furthercomprises processing the first set of identifiers and the second set ofidentifiers to determine an identifier purity for the single cellprocess.

In some embodiments, a given bead from the plurality of barcode beadscomprises a plurality of nucleic acid barcode molecules releasablyattached thereto. In some embodiments, the first nucleic acid barcodemolecule and the second nucleic acid barcode molecule is released fromthe first barcode bead and the second barcode bead.

In some embodiments, the plurality of barcode beads is a plurality ofgel beads.

In some embodiments, the nucleic acid molecule is releasably attached tothe control bead. In some embodiments, the nucleic acid molecule isreleased from the control bead.

In some embodiments, the nucleic acid molecule is within the controlbead.

In some embodiments, the control bead comprises a first functionalgroup, and said nucleic acid molecule comprising the known sequencecomprises a second functional group. In some embodiments, the nucleicacid molecule comprising the known sequence is attached to said controlbead by reacting said first functional group with said second functionalgroup. In some embodiments, the first functional group is an alkyne, atrans-cyclooctene, or an avidin, or any combination thereof. In someembodiments, the first functional group is an alkyne. In someembodiments, the second functional group is an azide, a tetrazine, or abiotin, or a combination thereof. In some embodiments, the secondfunctional group is an azide. In some embodiments, the first functionalgroup reacts with said second functional group in a click reaction. Insome embodiments, the click reaction is a copper-catalyzed azide-alkynecycloaddition reaction, an inverse-electron demand Diels-Alder reaction,or an avidin-biotin interaction. In some embodiments, the click reactionis a copper-catalyzed azide-alkyne cycloaddition reaction.

In some embodiments, said control bead is attached to said nucleic acidmolecule comprising the known sequence using bioconjugation chemistriesother than click chemistry. Thus, in some embodiments, said firstfunctional group is a carboxylic acid, and said second functional groupis an amine. Said first functional group can react with said secondfunctional group via an amide bond formation reaction. Such amide bondformation reaction can further comprise1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),N-hydroxysuccinimide (NETS), or a combination thereof.

In some embodiments, the known sequence is synthetic.

In some embodiments, the known sequence is derived from a biologicalsample.

In some embodiments, the control bead comprises a protein-DNA complexand the nucleic acid molecule is part of the protein-DNA complex. Insome embodiments, the known sequence comprises defined protein bindingsites. In some embodiments, the single cell process comprises ATAC-seq.

In some embodiments, the plurality of analyte carriers is a plurality ofcells. In some embodiments, the plurality of analyte carriers is aplurality of nuclei. In some embodiments, the plurality of analytecarriers is a plurality of cell beads.

In some embodiments, the plurality of partitions is a plurality ofdroplets. In some embodiments, the plurality of partitions is aplurality of wells.

In another aspect, provided is a kit for analyzing a cellular samplecomprising analyte carriers, comprising: a set of control beads, whereinat least a subset of said set of control beads comprise (i) identicalnucleic acid molecules each comprising a known nucleic acid sequence,and (ii) has a characteristic corresponding to within 70% deviation froman average value of said characteristic of said analyte carriers; and anindex comprising said known nucleic acid sequence.

In some embodiments, said characteristic is selected from a groupconsisting of size, shape, density, conductivity, hardness,deformability, and hydrophobicity.

In some embodiments, the index comprises a concentration of the knownnucleic acid sequence.

In some embodiments, the set of control beads comprises a first subsetof control beads and a second subset of control beads. In someembodiments, the first subset of control beads comprises a first set ofnucleic acid molecules comprising a first set of known sequences, andsaid second subset of control beads comprises a second set of nucleicacid molecules comprising a second set of known sequences. In someembodiments, the first set of known sequences comprises 96 differentknown sequences and said second set of known sequences comprises 96different known sequences. In some embodiments, the 96 different knownsequences of said first set of known sequences are different then said96 different known sequences of said second set of known sequences. Insome embodiments, the first subset of control beads and said secondsubset of control beads are present in a ratio. In some embodiments, theratio is at least about 1:0.001, 1:0.01, 1:0.1, 1:0.2, 1:0.3, 1:0.4,1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4,1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, or1:1000. In some embodiments, the ratio is at most about 1:1000, 1;100,1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7,1:6, 1:5, 1:4, 1:3, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4,1:1.3, 1:1.2, 1:1.1, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4,1:0.3, 1:0.2, 1:0.1, 1:0.01, or 1:0.001. In some embodiments, the ratiois about 1:2.

In some embodiments, a subset of the control beads comprises additionalidentical nucleic acid molecules that are different than the identicalnucleic acid molecules, each of which additional nucleic acid moleculescomprises an additional known nucleic acid sequence. In someembodiments, the index comprises the additional known nucleic acidsequence. In some embodiments, the index comprises a concentration ofthe additional known nucleic acid sequence. In some embodiments, theknown nucleic acid sequence and the additional known nucleic acidsequence vary in length, concentration, and/or GC content.

Another aspect of the present disclosure provides a non-transitorycomputer readable medium comprising machine executable code that, uponexecution by one or more computer processors, implements any of themethods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprisingone or more computer processors and computer memory coupled thereto. Thecomputer memory comprises machine executable code that, upon executionby the one or more computer processors, implements any of the methodsabove or elsewhere herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 shows an example of a microfluidic channel structure forpartitioning individual analyte carriers.

FIG. 2 shows an example of a microfluidic channel structure fordelivering barcode carrying beads to droplets.

FIG. 3 shows an example of a microfluidic channel structure forco-partitioning analyte carriers and reagents.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.FIG. 7B shows a perspective view of the channel structure of FIG. 7A.

FIG. 8 illustrates an example of a barcode carrying bead.

FIG. 9 illustrates a method for performing quality control for a singlecell experiment.

FIG. 10 illustrates two synthetic cells that may be used in specific,known ratios as sequencing standards.

FIG. 11 illustrates a HiScribe RNA synthesis with capping and tailingstarting from an RNA transcript. FIG. 11 discloses SEQ ID NOS 4-6,respectively, in order of appearance.

FIG. 12A shows digital droplet PCR results showing ligated(azide-containing) and un-ligated RNA sequences derived from a firsttranscript. FIG. 12B illustrates digital droplet PCR results showingligated (azide-containing) and un-ligated RNA sequences derived from asecond transcript.

FIG. 13 illustrates the use of click chemistry to attach RNA sequences(e.g., azide-functionalized) produced from RNA transcripts onto gelbeads (e.g., alkyne-functionalized) to generate a synthetic cell (e.g.,a control bead). The click reaction may be a Cu-mediated azide-alkynecycloaddition reaction. FIG. 13 discloses SEQ ID NOS 4-6, respectively,in order of appearance.

FIG. 14A shows a fluorescence spectrum for a control click reaction thatonly contains an alkyne but no azide and thus where no click reactionproducts were expected. FIG. 14B shows a right shift of the fluorescencesignal indicating an increase in fluorescence intensity confirming thatalkyne-functionalized gel beads were functionalized with azide-modifiedRNA molecules via Cu-mediated azide-alkyne click reaction in a firsttest reaction. FIG. 14C shows a right shift of the fluorescence signalindicating an increase in fluorescence intensity confirming thatalkyne-functionalized gel beads were functionalized with azide-modifiedRNA molecules via Cu-mediated azide-alkyne click reaction in a secondtest reaction. FIG. 14D shows a right shift of the fluorescence signalindicating an increase in fluorescence intensity confirming thatalkyne-functionalized gel beads were functionalized with azide-modifiedRNA molecules via Cu-mediated azide-alkyne click reaction in a thirdtest reaction.

FIG. 15 shows a diagram indicating signals from complementary DNA (cDNA)fragments that match the expected transcript sizes for both transcriptstested. The additional peak at about 75 bp may be due to a formed primerdimer.

FIG. 16 shows a bioanalyzer trace indicating that the expectedtranscripts were detected at approximately 311 bp and 674 bp. Theobserved peak width may be due to random fragmentation of the nucleicacid molecules.

FIG. 17 shows a computer system that is programmed or otherwiseconfigured to implement methods provided herein.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated. Ranges may be expressed herein as from“about” one particular value, and/or to “about” another particularvalue. When such a range is expressed, another possibility includes fromthe one particular value and/or to the other particular value.Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular value maybe another possibility. It will be further understood that the endpointsof each of the ranges are in relation to the other endpoint, andindependently of the other endpoint. The term “about” as used herein mayrefer to a range that is 15% plus or minus from a stated numerical valuewithin the context of the particular usage. For example, about 10 mayinclude a range from 8.5 to 11.5.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that conveys or is capable of conveying information about ananalyte. A barcode can be part of an analyte. A barcode can beindependent of an analyte. A barcode can be a tag attached to an analyte(e.g., nucleic acid molecule) or a combination of the tag in addition toan endogenous characteristic of the analyte (e.g., size of the analyteor end sequence(s)). A barcode may be unique. Barcodes can have avariety of different formats. For example, barcodes can include:polynucleotide barcodes; random nucleic acid and/or amino acidsequences; and synthetic nucleic acid and/or amino acid sequences. Abarcode can be attached to an analyte in a reversible or irreversiblemanner. A barcode can be added to, for example, a fragment of adeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before,during, and/or after sequencing of the sample. Barcodes can allow foridentification and/or quantification of individual sequencing-reads.

The term “real time,” as used herein, can refer to a response time ofless than about 1 second, a tenth of a second, a hundredth of a second,a millisecond, or less. The response time may be greater than 1 second.In some instances, real time can refer to simultaneous or substantiallysimultaneous processing, detection or identification.

The term “subject,” as used herein, generally refers to an animal, suchas a mammal (e.g., human) or avian (e.g., bird), or other organism, suchas a plant. For example, the subject can be a vertebrate, a mammal, arodent (e.g., a mouse), a primate, a simian or a human. Animals mayinclude, but are not limited to, farm animals, sport animals, and pets.A subject can be a healthy or asymptomatic individual, an individualthat has or is suspected of having a disease (e.g., cancer) or apre-disposition to the disease, and/or an individual that is in need oftherapy or suspected of needing therapy. A subject can be a patient. Asubject can be a microorganism or microbe (e.g., bacteria, fungi,archaea, viruses).

The term “genome,” as used herein, generally refers to genomicinformation from a subject, which may be, for example, at least aportion or an entirety of a subject's hereditary information. A genomecan be encoded either in DNA or in RNA. A genome can comprise codingregions (e.g., that code for proteins) as well as non-coding regions. Agenome can include the sequence of all chromosomes together in anorganism. For example, the human genome ordinarily has a total of 46chromosomes. The sequence of all of these together may constitute ahuman genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach, including ligation,hybridization, or other approaches.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example, nucleicacid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), including variants or derivatives thereof (e.g., single strandedDNA). Sequencing can be performed by various systems currentlyavailable, such as, without limitation, a sequencing system byIllumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or LifeTechnologies (Ion Torrent®). Alternatively or in addition, sequencingmay be performed using nucleic acid amplification, polymerase chainreaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR),or isothermal amplification. Such systems may provide a plurality of rawgenetic data corresponding to the genetic information of a subject(e.g., human), as generated by the systems from a sample provided by thesubject. In some examples, such systems provide sequencing reads (also“reads” herein). A read may include a string of nucleic acid basescorresponding to a sequence of a nucleic acid molecule that has beensequenced. In some situations, systems and methods provided herein maybe used with proteomic information.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel bead.The gel bead may include a polymer matrix (e.g., matrix formed bypolymerization or cross-linking). The polymer matrix may include one ormore polymers (e.g., polymers having different functional groups orrepeat units). Polymers in the polymer matrix may be randomly arranged,such as in random copolymers, and/or have ordered structures, such as inblock copolymers. Cross-linking can be via covalent, ionic, orinductive, interactions, or physical entanglement. The bead may be amacromolecule. The bead may be formed of nucleic acid molecules boundtogether. The bead may be formed via covalent or non-covalent assemblyof molecules (e.g., macromolecules), such as monomers or polymers. Suchpolymers or monomers may be natural or synthetic. Such polymers ormonomers may be or include, for example, nucleic acid molecules (e.g.,DNA or RNA). The bead may be formed of a polymeric material. The beadmay be magnetic or non-magnetic. The bead may be rigid. The bead may beflexible and/or compressible. The bead may be disruptable ordissolvable. The bead may be a solid particle (e.g., a metal-basedparticle including but not limited to iron oxide, gold or silver)covered with a coating comprising one or more polymers. Such coating maybe disruptable or dissolvable.

The terms “bead,” “gel bead,” and “synthetic cell,” may be usedinterchangeably herein and generally refer to a particle that can mimica cell. Such particle can be a bead such as a gel bead. The bead may besimilar to a cell, e.g., a cell of a biological sample in terms of size,shape, etc. A bead may be a control bead or a barcode bead. A controlbead may be structurally similar to a barcode bead. For example, in acontrol bead, at least some of the barcode sequence(s) of a barcode beadmay be replaced with control sequences (e.g., nucleic acid sequencessuch as genes). This modular design of control and/or barcode beads maybe advantageous and simplify production and/or quality control of suchbeads.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The biological sample may comprise any number ofmacromolecules, for example, cellular macromolecules. The sample may bea cell sample. The sample may be a cell line or cell culture sample. Thesample can include one or more cells. The sample can include one or moremicrobes. The biological sample may be a nucleic acid sample or proteinsample. The biological sample may also be a carbohydrate sample or alipid sample. The biological sample may be derived from another sample.The sample may be a tissue sample, such as a biopsy, core biopsy, needleaspirate, or fine needle aspirate. The sample may be a fluid sample,such as a blood sample, urine sample, or saliva sample. The sample maybe a skin sample. The sample may be a cheek swab. The sample may be aplasma or serum sample. The sample may be a cell-free or cell freesample. A cell-free sample may include extracellular polynucleotides.Extracellular polynucleotides may be isolated from a bodily sample thatmay be selected from the group consisting of blood, plasma, serum,urine, saliva, mucosal excretions, sputum, stool and tears.

The term “analyte carrier,” as used herein, generally refers to adiscrete biological system derived from a biological sample. The analytecarrier may be a macromolecule. The analyte carrier may be a smallmolecule. The analyte carrier may be a biological particle. The analytecarrier may be a virus. The analyte carrier may be a cell or derivativeof a cell. The analyte carrier may be an organelle. The analyte carriermay be a rare cell from a population of cells. The analyte carrier maybe any type of cell, including without limitation prokaryotic cells,eukaryotic cells, bacterial, fungal, plant, mammalian (e.g., from ahuman and/or a mouse), or other animal cell type, mycoplasmas, normaltissue cells, tumor cells, or any other cell type, whether derived fromsingle cell or multicellular organisms. The analyte carrier may be aconstituent of a cell. The analyte carrier may be or may include DNA,RNA, organelles, proteins, or any combination thereof. The analytecarrier may be or may include a matrix (e.g., a gel or polymer matrix)comprising a cell or one or more constituents from a cell (e.g., cellbead), such as DNA, RNA, organelles, proteins, or any combinationthereof, from the cell. The analyte carrier may be obtained from atissue of a subject. The analyte carrier may be a hardened cell. Suchhardened cell may or may not include a cell wall or cell membrane. Theanalyte carrier may include one or more constituents of a cell, but maynot include other constituents of the cell. An example of suchconstituents is a nucleus or an organelle. A cell may be a live cell.The live cell may be capable of being cultured, for example, beingcultured when enclosed in a gel or polymer matrix, or cultured whencomprising a gel or polymer matrix.

The term “macromolecular constituent,” as used herein, generally refersto a macromolecule contained within or from an analyte carrier. Themacromolecular constituent may comprise a nucleic acid. In some cases,the analyte carrier may be a macromolecule. The macromolecularconstituent may comprise DNA. The macromolecular constituent maycomprise RNA. The RNA may be coding or non-coding. The RNA may bemessenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), forexample. The RNA may be a transcript. The RNA may be small RNA that areless than 200 nucleic acid bases in length, or large RNA that aregreater than 200 nucleic acid bases in length. Small RNAs may include5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA(miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs),Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and smallrDNA-derived RNA (srRNA). The RNA may be double-stranded RNA orsingle-stranded RNA. The RNA may be circular RNA The macromolecularconstituent may comprise a protein. The macromolecular constituent maycomprise a peptide. The macromolecular constituent may comprise apolypeptide.

The term “molecular tag,” as used herein, generally refers to a moleculecapable of binding to a macromolecular constituent. The molecular tagmay bind to the macromolecular constituent with high affinity. Themolecular tag may bind to the macromolecular constituent with highspecificity. The molecular tag may comprise a nucleotide sequence. Themolecular tag may comprise a nucleic acid sequence. The nucleic acidsequence may be at least a portion or an entirety of the molecular tag.The molecular tag may be a nucleic acid molecule or may be part of anucleic acid molecule. The molecular tag may be an oligonucleotide or apolypeptide. The molecular tag may comprise a DNA aptamer. The moleculartag may be or comprise a primer. The molecular tag may be, or comprise,a protein. The molecular tag may comprise a polypeptide. The moleculartag may be a barcode.

The term “partition,” as used herein, generally, refers to a space orvolume that may be suitable to contain one or more species or conductone or more reactions. A partition may be a physical compartment, suchas a droplet or well. The partition may isolate space or volume fromanother space or volume. The droplet may be a first phase (e.g., aqueousphase) in a second phase (e.g., oil) immiscible with the first phase.The droplet may be a first phase in a second phase that does not phaseseparate from the first phase, such as, for example, a capsule orliposome in an aqueous phase. A partition may comprise one or more other(inner) partitions. In some cases, a partition may be a virtualcompartment that can be defined and identified by an index (e.g.,indexed libraries) across multiple and/or remote physical compartments.For example, a physical compartment may comprise a plurality of virtualcompartments.

Provided herein are systems and methods for analyzing a single cellprocess in a cell sample. Such systems and methods may provide syntheticcells that mimic single cell behavior in single cell processingexperiments. A synthetic cell may be a bead comprising one or more knownsequences. For example, the bead may comprise a nucleic acid molecule(e.g., DNA, RNA, etc.) comprising a known sequence. The bead may becoupled to, or otherwise incorporate, the nucleic acid molecule. In someinstances, the nucleic acid molecule may be captured by the bead (e.g.,in a matrix). The bead may be a gel bead. The synthetic cell may beintroduced into a cell sample and have approximately the same orsubstantially the same size as other cells in the cell sample (e.g.,from about 15 micrometers (μm) to about 60 μm). The synthetic cell maybe carried through the entire workflow of a single cell processingexperiment with the cell sample. After sequencing, the one or more knownsequences in the synthetic cell may be identified and analyzed todetermine various characteristics of the single cell processingexperiment, such as the effectiveness or the efficiency of the librarypreparation process or the sequencing process.

Quality Control for Single Cell Processing

In an aspect, the present disclosure provides a method for analyzing asingle cell process in a cell sample by distributing a set of barcodebeads, a set of control beads, and the cell sample into a plurality ofpartitions. Systems and methods for generating partitions are describedelsewhere herein, such as with respect to FIGS. 1-7B. The partitions maybe droplets in emulsions or other microcapsules. The partitions may besolid partitions, such as wells. Barcode beads are described elsewhereherein, such as with respect to FIG. 8. The systems and methods may usemore than one set of control beads. For example, there may be at leastabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more sets of control beads.Alternatively, there may be at most about 20, 15, 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 set of control beads.

The method may comprise generating the plurality of partitions includinga first partition and a second partition. The first partition maycomprise an analyte carrier, such as a cell, from the cell sample and afirst barcode bead from the set of barcode beads. The analyte carriermay be or comprise a biological particle. The analyte carrier may be orcomprise an analyte. The analyte carrier may comprise a template nucleicacid molecule. The first barcode bead may comprise a first barcodenucleic acid molecule comprising a first barcode sequence. The secondpartition may comprise a second barcode bead from the set of barcodebeads and a first control bead from the set of control beads. The secondbarcode bead may comprise a second barcode nucleic acid moleculecomprising a second barcode sequence. The second barcode sequence may bedifferent from the first barcode sequence. The first control bead maycomprise a nucleic acid molecule comprising a known sequence.

The method may comprise processing the first partition and the secondpartition to generate a barcoded template nucleic acid molecule and abarcoded nucleic acid molecule. The barcoded template nucleic acidmolecule may comprise the first barcode sequence to identify the analytecarrier (e.g., cell). The barcoded nucleic acid molecule may comprisethe second barcode sequence to identify the known sequence. The barcodedtemplate nucleic acid molecule and the barcoded nucleic acid moleculemay be sequenced to identify the analyte carrier and/or the knownsequence to analyze the single cell process.

As described elsewhere herein, in some cases, the method may furthercomprise using a second set of control beads. For example, a thirdpartition may be generated. The third partition comprises a thirdbarcode bead from the set of barcode beads and a second control beadfrom the second set of control beads. The third barcode bead maycomprise a third barcode nucleic acid molecule comprising a thirdbarcode sequence. The second control bead may comprise a second nucleicacid molecule comprising a second known sequence. The second knownsequence may be different from the known sequence. The third partitionmay be processed to generate a second barcoded nucleic acid molecule.The second barcoded nucleic acid molecule may comprise the third barcodesequence to identify the second known sequence. The second barcodednucleic acid molecule may be sequenced, in addition to the barcodedtemplate nucleic acid molecule and the barcoded nucleic acid molecule toanalyze the single cell process.

FIG. 9 illustrates a method for performing quality control for a singlecell experiment. A mixed biological sample 902 and a set of barcodebeads 904 may be partitioned into a plurality of partitions 906. Themixed biological sample 902 may comprise a plurality of cells 908, aplurality of first control beads 910, and a plurality of second controlbeads 912. The set of barcode beads 904 may comprise a plurality ofbarcode beads 914. Each partition in the plurality of partitions 906 maycomprise a barcode bead and one cell of the plurality of cells of thebiological sample, a first control bead, or a second control bead. Forexample, some partitions 916 may comprise a barcode bead and a cell,some partitions 918 may comprise a barcode bead and a first controlbead, and some partitions 920 may comprise a barcode bead and a secondcontrol bead. It will be appreciated, as described elsewhere herein,that during partition generation, a partition may have no particles(e.g., beads, cells, etc.), have more than one barcode beads or morethan one of a cell, a first control bead, or a second control bead, orany combination thereof. Systems and methods may be configured togenerate mostly singularly-occupied partitions (e.g., greater than 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or higher, etc.) and/orconfigured to sort out singularly-occupied partitions for furtherprocessing. Singularly-occupied partitions may refer to those partitionshaving one of a barcode bead and/or one of a cell or control bead (e.g.,first control bead, second control bead).

A barcode bead (e.g., 914) in the set of barcode beads 914 may comprisea barcode. An example of a barcode carrying bead is shown in FIG. 8. Thebead may be coupled to a nucleic acid molecule, such as anoligonucleotide, by a releasable linkage (e.g., disulfide linker). Thebead may be coupled to one or more other nucleic acid molecules. Eachnucleic acid molecule may be or comprise a bead-specific barcodesequence (e.g., unique to each bead) that is common to all nucleic acidmolecules coupled to the same bead. The barcode may comprise a number ofsequence elements, such as the bead-specific barcode sequence, afunctional sequence used for subsequent processing (e.g., flow cellattachment sequence, sequencing primer sequence, etc.), a primingsequence (e.g., poly-T sequence, targeted priming sequence, randompriming sequence, etc.), an anchoring sequence (e.g., random shortsequences, etc.), and/or a molecule-specific barcode sequence. Themolecule-specific barcode sequence, also referred to as a uniquemolecular identifier (UMI), may be unique to each nucleic acid moleculecoupled to the bead, even within the same bead. For example, for abarcode bead coupled to tens to hundreds of thousands or even millionsof individual nucleic acid molecules, each individual nucleic acidmolecule may be uniquely identified by the UMI. As will be appreciated,two or more UMIs may overlap. For example, the UMI count purity maymeasure chimerism in the PCR which contributes to significant technicalnoise at higher sequencing depths. Beneficially, the systems and methodsdescribed herein may determine a UMI purity rate for the single cellexperiment.

A first control bead from the plurality of first control beads 910 maybe a synthetic cell configured to mimic single cell behavior for asingle cell processing experiment, such as for sequencing. The firstcontrol bead may be a bead, such as a gel bead. The first control beadmay have approximately the same or substantially the same size as theaverage size of the plurality of cells 908 in the mixed biologicalsample 902. For example, the first control bead may have a diameter fromabout 15 micrometers (μm) to about 25 μm. Alternatively or in additionto, the first control bead may have a diameter from at least about 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 65 μm or greater. Alternatively or inaddition to, the first control bead may have a diameter of at most about65, 60, 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10 μm or less. In some instances,the size (e.g., diameter) of a first control bead may deviate from anaverage size (e.g., diameter) of a cell in the mixed cell sample withinat least about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or lower. Alternatively or inaddition to, the first control bead may have other physicalcharacteristics or properties that mimic a single cell, such as density,mass, shape, and the like. Alternatively or in addition to, the firstcontrol bead may have other characteristics or properties, such aschemical properties, that mimic a single cell, such as conductivity,hardness, deformability, interactive properties with aqueous ornon-aqueous solutions, and the like. In some instances, the firstcontrol bead may have optical properties that mimic a single cell, suchas those that are discernable with flow cytometry (e.g., forward or sidescatter).

The first control bead may comprise a first set of one or more knownsequences. For example, the bead may comprise a nucleic acid molecule(e.g., DNA, RNA, etc.) comprising a known sequence. The first controlbead may comprise any other analyte (e.g., proteins, metabolite, nucleicacid molecule, etc.) comprising a known sequence. In some instances, thebead may be coupled to, or otherwise incorporate, the nucleic acidmolecule (or other analyte). For example, the bead may be releasablycoupled to the nucleic acid molecule, such as with a linker or withother methods described elsewhere herein. In some instances, the beadmay be coupled to or attached to the nucleic acid molecule using clickchemistry as described elsewhere herein. In some instances, the nucleicacid molecule (or other analyte) may be captured by the bead (e.g., in apolymer matrix). The first control bead may comprise any number of knownsequences. For example, the first bead control bead may comprise atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, or 100 or more knownsequences. Alternatively or in addition to, the first bead control beadmay comprise at most about 100, 96, 95, 90, 85, 80, 75, 70, 65, 60, 55,50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 knownsequences. The first control bead may comprise as many and/or anycombination of different analytes (e.g., nucleic acid molecules). In anexample, the first control bead may comprise a first DNA moleculecomprising a first known sequence, a second DNA molecule comprising asecond known sequence, and a RNA molecule comprising a third knownsequence. The first control bead may comprise one or more additionalanalytes that do not comprise a known sequence. For example, suchanalytes may allow the first control bead to mimic single cell behavior.

A second control bead from the plurality of second control beads 912 maybe another synthetic cell, such as described with respect to the firstcontrol bead. The second control bead may comprise a second set of oneor more known sequences. For example, the bead may comprise a nucleicacid molecule (e.g., DNA, RNA, etc.) or other analyte (e.g., proteins,metabolite, nucleic acid molecule, etc.) comprising a known sequence. Insome instances, the second control bead may be coupled to, or otherwiseincorporate, the nucleic acid molecule (or other analyte). For example,the second control bead may be releasably coupled to the nucleic acidmolecule, such as with a linker or with other methods describedelsewhere herein. In some instances, the bead may be coupled to orattached to the nucleic acid molecule using click chemistry as describedelsewhere herein. In some instances, the nucleic acid molecule (or otheranalyte) may be captured by the bead (e.g., in a polymer matrix). Thesecond control bead may comprise any number of known sequences. Forexample, the second control bead may comprise at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 96, 100 or more known sequences. Alternatively or inaddition to, the second control bead may comprise at most about 100, 96,95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10,9, 8, 7, 6, 5, 4, 3, 2, or 1 known sequences. The second control beadmay comprise as many and/or any combination of different analytes (e.g.,nucleic acid molecules). In an example, the second control bead maycomprise a first DNA molecule comprising a first known sequence, asecond DNA molecule comprising a second known sequence, and a RNAmolecule comprising a third known sequence. The second control bead maycomprise one or more additional analytes that do not comprise a knownsequence. For example, such analytes may allow the second control beadto mimic single cell behavior.

The first set of known sequences in the first control bead may bedifferent from the second set of known sequences in the second controlbead. For example, they may be mutually exclusive, such that none of theone or more known sequences in the second control bead overlaps with anyof the one or more known sequences in the first control bead. In someinstances, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, 99.9% or more transcripts in the first set of knownsequences may be separate from and independently classifiable from thesecond set of known sequences. Alternatively or in addition, at mostabout 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90% orless transcripts in the first set of known sequences may be separatefrom and independently classifiable from the second set of knownsequences. In other instances, there may be one or more overlappingknown sequences.

In some instances, each set of known sequences may comprise a unique setof sequences. For example, the unique set of sequences may vary inlength, concentration, and/or GC content.

In some instances, the known sequences may be synthetic. In otherinstances, the known sequences may be derived from a biological sample.Alternatively or in addition to, the known sequences may be acombination of synthetic and biologically derived sequences. In someinstances, a first set of known sequences for the first control bead maybe derived from a first species (e.g., purified genomic DNA from ahuman), and a second set of known sequences for the second control beadmay be derived from a second species (e.g., purified genomic DNA from amouse). In some instances, the sources of the known sequences used inthe control beads may vary depending on the type of single cellapplication, type of parameter (e.g., UMI count purity, doublet rate,etc.) that is to be determined, and/or method of determination. Forexample, laboratories may use readily available, well-characterized celllines to provide the known sequences as control. But using such controlsmay yield variable results depending on a variety of biological factors,and it may be difficult (or in some cases impossible) to calculatedoublet rate or UMI purity. In another example, a first set of knownsequences may be derived from the peripheral blood mononuclear cells(PBMCs) from a first human donor and a second set of known sequences maybe derived from the PBMCs from a second human donor, and the singlenucleotide polymorphisms (SNPs) may be analyzed to determine the doubletrate, but not the UMI purity. In another example, a first set of knownsequences may be derived from the peripheral blood mononuclear cells(PBMCs) from a female human and a second set of known sequences may bederived from the PBMCs from a male human, and the Y chromosomes may beanalyzed to determine the doublet rate, but not the UMI purity.

The systems and methods may comprise any ratio of the number orconcentration of the plurality of first control beads 910 to theplurality of second control beads 912. For example, the ratio may be1:1. Alternatively or in addition to, the ratio may be at least about1:0.001, 1:0.01, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8,1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8,1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40,1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:1000 or more. Alternatively or inaddition to, the ratio may be at most about 1:1000, 1;100, 1:90, 1:80,1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4,1:3, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1,1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, 1:0.1,1:0.01, 1:0.001 or less.

Methods for generating a bead are described in detail elsewhere herein.A control bead may be generated as a gel bead, for example,encapsulating one or more nucleic acid molecules comprising the knownsequences in the control bead. For example, nucleic acid moleculescomprising the known sequences may be added into the starting reagentmix (e.g., instead of acrydite primers). The control bead may beconfigured to have smaller pore size, such as by increasing cross-linkerconcentration. For example, the pores may prevent DNA molecules, RNAmolecules, or other analytes from escaping the control bead. Inoperation, the control beads may be packaged into correct buffers at aknown concentration, and stored in single use aliquots. The controlbeads may be stored at ambient temperatures. The control beads may bestored at room temperatures. In some instances, the control beads may bestored at about −80 degrees Celsius (° C.). Alternatively or in additionto, the control beads may be stored at (or otherwise exposed to)temperatures of at most about 25° C., 24° C., 23° C., 22° C., 21° C.,20° C., 19° C., 18° C., 17° C., 16° C., 15° C., 14° C., 13° C., 12° C.,11° C., 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., 4° C., 3° C., 2° C.,1° C., 0° C., −5° C., −10° C., −15° C., −20° C., −25° C., −30° C., −35°C., −40° C., −45° C., −50° C., −55° C., −60° C., −65° C., −70° C., −75°C., −80° C. or less. In some instances, the control beads may be storedat (or otherwise exposed to) temperatures of more than about 25° C.

Alternatively or in addition to, control beads of the present disclosuremay be generated as non-gel beads, such as polystyrene beads,encapsulating one or more nucleic acid molecules comprising the knownsequences.

In operation, as illustrated in FIG. 9, a plurality of first controlbeads 910 and a plurality of second control beads 912 may be introducedinto a cell sample comprising a plurality of cells 908 to generate amixed cell sample 902. Any number of each type of control bead may beintroduced into the cell sample. For example, at least about 2, 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 200, 300, 400, 500, 600, 700, 800 or more control beads may beintroduced. Alternatively or in addition to, at most about 800, 700,600, 500, 400, 300, 200, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60,50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 control bead may beintroduced into the cell sample. Alternatively or in addition to, aratio of a first concentration of first control beads and/or secondcontrol beads to a second concentration of cells in the mixed cellsample may be at least about 1:1000, 1;100, 1:90, 1:80, 1:70, 1:60,1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2,1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2, 1:1.1, 1:1,1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, 1:0.1, 1:0.01,1:0.001 or more. Alternatively or in addition to, the ratio may be atmost about 1:0.001, 1:0.01, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6,1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20,1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:1000 or less. Themixed cell sample 902 may be partitioned with the set of barcode beads904 to generate the plurality of partitions 906. Each partition in theplurality of partitions 906 may comprise a barcode bead and one of acell, a first control bead, or a second control bead. For example, somepartitions 916 may comprise a barcode bead and a cell, some partitions918 may comprise a barcode bead and a first control bead, and somepartitions 920 may comprise a barcode bead and a second control bead.Libraries may be created and sequenced from the plurality of partitions906.

For example, in a first partition (e.g., 916), a first cell (e.g., 908)is co-partitioned with a first barcode bead (e.g., 914). The firstbarcode bead may comprise one or more barcode nucleic acid molecules,each individual barcode nucleic acid molecule (i) sharing a common,bead-specific, first barcode sequence and (ii) having a different uniquemolecular identifier (UMI). In some instances, the first cell may belysed in the partition (e.g., via co-partitioning with a lysing agent)to release one or more analyte carriers. The one or more analytecarriers may comprise template nucleic acid molecules, such as DNA, RNA,and the like. In the partition, the barcode nucleic acid molecules canbe released from the first barcode bead to hybridize to the analytecarriers. By way of example, where an analyte carrier is an mRNAmolecule, a poly-T segment of the barcode nucleic acid molecule canhybridize to the poly-A tail of the mRNA molecule to generate a barcodedmRNA molecule. Each analyte carrier from the first cell may behybridized to a different barcode nucleic acid molecule, thereby eachbeing labeled by the first barcode sequence and a different UMI. Forexample, a barcoded template nucleic acid molecule may be generated.

In a second partition (e.g., 918), a first control bead (e.g., 910) isco-partitioned with a second barcode bead (e.g., 914). The secondbarcode bead may comprise one or more barcode nucleic acid molecules,each individual barcode nucleic acid molecule (i) sharing a common,bead-specific, second barcode sequence and (ii) having a differentunique molecular identifier (UMI). The first control bead may comprise aplurality of analyte carriers. By way of example, the analyte carriersmay comprise a first nucleic acid molecule with a first known sequenceand a second nucleic acid molecule with a second known sequence. In someinstances, the first control bead may be stimulated to release the firstnucleic acid molecule and the second nucleic acid molecule from thefirst control bead. In the partition, the barcode nucleic acid moleculescan be released from the second barcode bead to hybridize to the analytecarriers of the first control bead and generate barcoded nucleic acidmolecules. Each analyte carrier from the first control bead may behybridized to a different barcode nucleic acid molecule, thereby eachbeing labeled by the second barcode sequence and a different UMI. Forexample, a first barcoded nucleic acid molecule and a second barcodednucleic acid molecule may be generated. The first barcoded nucleic acidmolecule may comprise the first known sequence, the second barcodesequence, and a UMI. The second barcoded nucleic acid molecule maycomprise the second known sequence, the second barcode sequence, and aUMI.

In a third partition (e.g., 920), a second control bead (e.g., 912) isco-partitioned with a third barcode bead (e.g., 914). The third barcodebead may comprise one or more barcode nucleic acid molecules, eachindividual barcode nucleic acid molecule (i) sharing a common,bead-specific, third barcode sequence and (ii) having a different uniquemolecular identifier (UMI). The second control bead may comprise aplurality of analyte carriers. By way of example, the analyte carriersmay comprise a third nucleic acid molecule with a third known sequenceand a fourth nucleic acid molecule with a fourth known sequence. In someinstances, the second control bead may be stimulated to release thethird nucleic acid molecule and the fourth nucleic acid molecule fromthe second control bead. In the partition, the barcode nucleic acidmolecules can be released from the third barcode bead to hybridize tothe analyte carriers of the second control bead and generate barcodednucleic acid molecules. Each analyte carrier from the second controlbead may be hybridized to a different barcode nucleic acid molecule,thereby each being labeled by the third barcode sequence and a differentUMI. For example, a third barcoded nucleic acid molecule and a fourthbarcoded nucleic acid molecule may be generated. The third barcodednucleic acid molecule may comprise the third known sequence, the thirdbarcode sequence, and a UMI. The fourth barcoded nucleic acid moleculemay comprise the fourth known sequence, the third barcode sequence, anda UMI.

The barcoded nucleic acid molecules from the plurality of partitions 906may be sequenced. In the above example, from the sequencing results, thefirst barcode sequence may be identified to identify the first cell, thesecond barcode sequence may be identified to identify the first controlbead, and the third barcode sequence may be identified to identify thesecond control bead. From the sequencing results, the first knownsequence may be identified to identify the first nucleic acid moleculeand the first control bead, the second known sequence may be identifiedto identify the second nucleic acid molecule and the first control bead,the third known sequence may be identified to identify the third nucleicacid molecule and the second control bead, and the fourth known sequencemay be identified to identify the fourth nucleic acid molecule and thesecond control bead.

During the single cell process, each cell or control bead in the mixedcell sample 902 is barcoded by a unique bead-specific barcode sequence,such that each analyte having the same bead-specific barcode sequencemay be attributed to the same cell or control bead. However, a doubletsituation may arise where two or more cells and/or control beads arebarcoded by the same bead-specific sequence. For example, where a firstcell and a second cell are barcoded by the same bead-specific barcodesequence, two analytes that respectively originated from the first celland the second cell, and having been barcoded by the same bead-specificbarcode sequence, may incorrectly be attributed as having originatedfrom the same cell. For example, a doublet situation may be caused bytwo or more beads in the set of beads 904 having the same bead-specificbarcode sequence or where a partition co-partitions one bead with two ormore cells and/or control beads (which is more likely). An example ofthe latter situation is illustrated in partition 920. Beneficially, thesystems and methods described herein may measure doublet rate. Doubletrate may be measured by comparing the barcode sequence on the knownsequences in the first control bead (e.g., first known sequence, secondknown sequence, etc.) with the barcode sequence on the known sequencesin the second control bead (e.g., third known sequence, fourth knownsequence, etc.). Any overlap may be indicative of doublets. Doublet ratemay be measured based at least in part on the number of overlappinginstances and the concentration of the first control beads 910 and/orthe second control beads 912 in the mixed cell sample 902. For clarity,for the purpose of measuring or investigating doublet rate, the presenceof UMIs is not required. That is, the barcode bead need not containdifferent UMIs on individual barcode nucleic acid molecules (i.e., theirpresence or absence is optional). For example, a barcode bead used inthe systems and methods described herein may comprise one or morebarcode nucleic acid molecules, each individual barcode nucleic acidmolecule sharing a common, bead-specific, barcode sequence.

In some instances, each analyte may be barcoded by a unique molecularidentifier. Ideally, during the single cell process, each analyte in acell or control bead is barcoded by a truly unique molecular identifier,such that each analyte has a different unique molecular identifier.Beneficially, identical copies of analytes arising from distinct cellsmay be distinguished from the identical copies arising fromamplification (e.g., PCR). Even following any subsequent amplificationof the contents of a given partition, the number of different UMIs canbe indicative of the quantity of the analyte originating from a givenpartition, and thus from the analyte carrier (e.g., cell). Such UMIcounts may be used to compare, for example, the number of copies of eachtranscript in different cell types or conditions. However, there may beerrors in a UMI count when, for example, foreign UMIs are introducedduring the single cell process. For example, a UMI sequence may bechanged when there are nucleotide substitutions during PCR or nucleotidemiscalling, or insertions or deletions (indels) during sequencing. Forexample, such changes may be caused by recombination events during PCRthat create chimeric sequences that can change an original UMI sequence.Beneficially, the systems and methods described herein may measure UMIpurity. Beneficially, the systems and methods described herein maymeasure both doublet rate and UMI purity in the same assay. In the aboveexample, UMI purity may be measured by comparing the UMI count for thefirst control bead to an expected UMI count based on the known sequencesin the first control bead (e.g., first known sequence, second knownsequence, etc.). Alternatively or in addition to, UMI purity may bemeasured by comparing the UMI count for the second control bead to anexpected UMI count based on the known sequences in the second controlbead (e.g., third known sequence, fourth known sequence, etc.). UMIpurity may be measured based at least in part on the concentration ofthe first control beads 910 and/or the second control beads 912 in themixed cell sample 902.

As described elsewhere herein, the systems and methods may use anynumber of types of control beads. For example, there may be at leastabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more sets of control beads.Alternatively, there may be at most about 20, 15, 10, 9, 8, 7, 6, 5, 4,3, 2, or 1 set of control beads. Such multiple sets of control beads maybe introduced into the cell sample at any ratio.

The systems and methods described herein may be used to perform qualitycontrol on any single cell assay or process thereof, such as apartitioning process, barcoding process, amplification process, librarycreation and sequencing process, and other processes (or combinationsthereof).

In some instances, a control bead may be configured to mimic differentanalytes. For example, a control bead of the present disclosure may beconfigured to mimic cell beads containing cells for analyzing singlecell applications utilizing cell beads. Cell beads are described indetail elsewhere herein, such as with respect to FIG. 1. For example thecontrol bead may be about 54 μm in diameter to mimic a cell bead.Alternatively or in addition to, the control bead may have a diameterfrom at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65 μm orgreater. Alternatively or in addition to, the control bead may have adiameter of at most about 65, 60, 55, 50, 45, 40, 35, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 μm orless. In some instances, the size (e.g., diameter) of the control beadmay deviate from an average size (e.g., diameter) of a cell bead in amixed cell bead sample (e.g., mixing a plurality of cell beads with thecontrol bead(s)) within at least about 90%, 80%, 70%, 60%, 50%, 40%,30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or lower.Alternatively or in addition to, the control bead may have otherphysical characteristics or properties that mimic a cell bead, such asdensity, mass, shape, and the like. The control bead may have othercharacteristics or properties, such as chemical properties, that mimic acell bead, such as conductivity, hardness, size, shape, density,hydrophobicity, and/or interactive properties with aqueous ornon-aqueous solutions, and the like. In operation, the control beads maybe introduced to a cell bead sample, and the mixed cell bead sample maybe subjected to the single cell application or assay. The control beads,or known sequences thereof, may be tracked during or after the singlecell application or assay for analysis.

In some instances, a control bead may be configured to mimic, orcomprise, a chromatin. For example, nucleic acid fragments may begenerated (e.g., synthetic or biologically derived) to have definedprotein binding sites as well as open stretches. The control bead maycomprise a protein-DNA molecule complex, wherein the DNA molecule in thecomplex comprises a known sequence. The known sequence may comprise thedefined protein binding sites. The control bead may be mixed with otheranalytes comprising protein-DNA molecule complexes for use in singlecell applications, such as an Assay for Transposase Accessible Chromatin(ATAC-seq). The control bead may be configured to mimic, or comprise,any other analyte (e.g., cell, cell bead, chromatin, nucleic acidmolecule, protein, another molecule, a complex, etc.) for use ascontrols in other single cell applications. The control beads, or knownsequences thereof, may be tracked during or after the single cellapplication or assay for analysis.

Kits

The present disclosure provides kits for use with methods and systemsdescribed herein. A kit can include a set of control beads. The set ofcontrol beads may be any set of control beads described elsewhereherein. For example, the set of control beads may be configured to mimiccells, cell beads, or other analytes. The kit may include an indexcomprising the set of known nucleic acid sequences in the set of controlbeads. For example, the index may be list of the known nucleic acidsequences. In some instances, the index may comprise the concentrationof each known sequence. In some instances, the index may comprise theconcentration of the set of control beads in a buffer. In someinstances, the index may comprise the concentration of any otherreagents included in the kit. The kit can include reagents and buffersnecessary for performing the methods described herein. For example, thekit can include reagents and buffers for sample preparation for asequencing assay and/or reagents and buffers for performing one or moresequencing assays described herein.

The kit may comprise any number of sets of control beads, such asdescribed elsewhere herein. For example, the kit may comprise at leastabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more sets of controlbeads. Alternatively or in addition to, the kit may comprise at mostabout 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 set of controlbeads. Such set(s) (or subset(s)) of control beads may be used in a kitat any ratio (e.g., in a 1:2 ratio). The index may comprise the sets ofknown nucleic acid sequences for each set of control beads included inthe kit. The index may comprise the concentration of each knownsequence. The index may comprise the concentration of each set ofcontrol beads in a respective buffer.

The kit may comprise instructions detailing one or more processes of thesystems and methods described herein. The kit can include a carrier,package, or container that may be compartmentalized to receive one ormore containers, such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements, such as thenucleic acid probes and buffers, to be used in a method describedherein. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers can be formed from a variety ofmaterials such as glass or plastic. The kit may include any combinationof the above. A kit may include all of the above. The articles ofmanufacture provided herein contain packaging materials. Examples ofpackaging materials include, but are not limited to, bottles, tubes,bags, containers, or bottles. A kit can include labels listing contentsof the kit and/or instructions for use, and package inserts withinstructions for use. A set of instructions can also be included. Theinstructions may be in physical or digital format (e.g., instructionsthat may be included in a pamphlet or stored in computer memory).

Systems and Methods for Sample Compartmentalization

In an aspect, the systems and methods described herein provide for thecompartmentalization, depositing, or partitioning of one or moreparticles (e.g., analyte carriers, macromolecular constituents ofanalyte carriers, beads, reagents, etc.) into discrete compartments orpartitions (referred to interchangeably herein as partitions), whereeach partition maintains separation of its own contents from thecontents of other partitions. The partition can be a droplet in anemulsion. A partition may comprise one or more other partitions.

A partition may include one or more particles. A partition may includeone or more types of particles. For example, a partition of the presentdisclosure may comprise one or more analyte carriers and/ormacromolecular constituents thereof. A partition may comprise one ormore gel beads. A partition may comprise one or more cell beads. Apartition may include a single gel bead, a single cell bead, or both asingle cell bead and single gel bead. A partition may include one ormore reagents. Alternatively, a partition may be unoccupied. Forexample, a partition may not comprise a bead. A cell bead can be ananalyte carrier and/or one or more of its macromolecular constituentsencased inside of a gel or polymer matrix, such as via polymerization ofa droplet containing the analyte carrier and precursors capable of beingpolymerized or gelled. Unique identifiers, such as barcodes, may beinjected into the droplets previous to, subsequent to, or concurrentlywith droplet generation, such as via a microcapsule (e.g., bead), asdescribed elsewhere herein. Microfluidic channel networks (e.g., on achip) can be utilized to generate partitions as described herein.Alternative mechanisms may also be employed in the partitioning ofindividual analyte carriers, including porous membranes through whichaqueous mixtures of cells are extruded into non-aqueous fluids.

The partitions can be flowable within fluid streams. The partitions maycomprise, for example, micro-vesicles that have an outer barriersurrounding an inner fluid center or core. In some cases, the partitionsmay comprise a porous matrix that is capable of entraining and/orretaining materials within its matrix. The partitions can be droplets ofa first phase within a second phase, wherein the first and second phasesare immiscible. For example, the partitions can be droplets of aqueousfluid within a non-aqueous continuous phase (e.g., oil phase). Inanother example, the partitions can be droplets of a non-aqueous fluidwithin an aqueous phase. In some examples, the partitions may beprovided in a water-in-oil emulsion or oil-in-water emulsion. A varietyof different vessels are described in, for example, U.S. PatentApplication Publication No. 2014/0155295, which is entirely incorporatedherein by reference for all purposes. Emulsion systems for creatingstable droplets in non-aqueous or oil continuous phases are describedin, for example, U.S. Patent Application Publication No. 2010/0105112,which is entirely incorporated herein by reference for all purposes.

In the case of droplets in an emulsion, allocating individual particlesto discrete partitions may in one non-limiting example be accomplishedby introducing a flowing stream of particles in an aqueous fluid into aflowing stream of a non-aqueous fluid, such that droplets are generatedat the junction of the two streams. Fluid properties (e.g., fluid flowrates, fluid viscosities, etc.), particle properties (e.g., volumefraction, particle size, particle concentration, etc.), microfluidicarchitectures (e.g., channel geometry, etc.), and other parameters maybe adjusted to control the occupancy of the resulting partitions (e.g.,number of analyte carriers per partition, number of beads per partition,etc.). For example, partition occupancy can be controlled by providingthe aqueous stream at a certain concentration and/or flow rate ofparticles. To generate single analyte carrier partitions, the relativeflow rates of the immiscible fluids can be selected such that, onaverage, the partitions may contain less than one analyte carrier perpartition in order to ensure that those partitions that are occupied areprimarily singly occupied. In some cases, partitions among a pluralityof partitions may contain at most one analyte carrier (e.g., bead, DNA,cell or cellular material). In some embodiments, the various parameters(e.g., fluid properties, particle properties, microfluidicarchitectures, etc.) may be selected or adjusted such that a majority ofpartitions are occupied, for example, allowing for only a smallpercentage of unoccupied partitions. The flows and channel architecturescan be controlled as to ensure a given number of singly occupiedpartitions, less than a certain level of unoccupied partitions and/orless than a certain level of multiply occupied partitions.

FIG. 1 shows an example of a microfluidic channel structure 100 forpartitioning individual analyte carriers. The channel structure 100 caninclude channel segments 102, 104, 106 and 108 communicating at achannel junction 110. In operation, a first aqueous fluid 112 thatincludes suspended analyte carriers (or cells) 114 may be transportedalong channel segment 102 into junction 110, while a second fluid 116that is immiscible with the aqueous fluid 112 is delivered to thejunction 110 from each of channel segments 104 and 106 to creatediscrete droplets 118, 120 of the first aqueous fluid 112 flowing intochannel segment 108, and flowing away from junction 110. The channelsegment 108 may be fluidically coupled to an outlet reservoir where thediscrete droplets can be stored and/or harvested. A discrete dropletgenerated may include an individual analyte carrier 114 (such asdroplets 118). A discrete droplet generated may include more than oneindividual analyte carrier 114 (not shown in FIG. 1). A discrete dropletmay contain no analyte carrier 114 (such as droplet 120). Each discretepartition may maintain separation of its own contents (e.g., individualanalyte carrier 114) from the contents of other partitions.

The second fluid 116 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets118, 120. Examples of particularly useful partitioning fluids andfluorosurfactants are described, for example, in U.S. Patent ApplicationPublication No. 2010/0105112, which is entirely incorporated herein byreference for all purposes.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 100 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junction.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying particles (e.g., analyte carriers, cellbeads, and/or gel beads) that meet at a channel junction. Fluid may bedirected to flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

The generated droplets may comprise two subsets of droplets: (1)occupied droplets 118, containing one or more analyte carriers 114, and(2) unoccupied droplets 120, not containing any analyte carriers 114.Occupied droplets 118 may comprise singly occupied droplets (having oneanalyte carrier) and multiply occupied droplets (having more than oneanalyte carrier). As described elsewhere herein, in some cases, themajority of occupied partitions can include no more than one analytecarrier per occupied partition and some of the generated partitions canbe unoccupied (of any analyte carrier). In some cases, though, some ofthe occupied partitions may include more than one analyte carrier. Insome cases, the partitioning process may be controlled such that fewerthan about 25% of the occupied partitions contain more than one analytecarrier, and in many cases, fewer than about 20% of the occupiedpartitions have more than one analyte carrier, while in some cases,fewer than about 10% or even fewer than about 5% of the occupiedpartitions include more than one analyte carrier per partition.

In some cases, it may be desirable to minimize the creation of excessivenumbers of empty partitions, such as to reduce costs and/or increaseefficiency. While this minimization may be achieved by providing asufficient number of analyte carriers (e.g., analyte carriers 114) atthe partitioning junction 110, such as to ensure that at least oneanalyte carrier is encapsulated in a partition, the Poissoniandistribution may expectedly increase the number of partitions thatinclude multiple analyte carriers. As such, where singly occupiedpartitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% orless of the generated partitions can be unoccupied.

In some cases, the flow of one or more of the analyte carriers (e.g., inchannel segment 102), or other fluids directed into the partitioningjunction (e.g., in channel segments 104, 106) can be controlled suchthat, in many cases, no more than about 50% of the generated partitions,no more than about 25% of the generated partitions, or no more thanabout 10% of the generated partitions are unoccupied. These flows can becontrolled so as to present a non-Poissonian distribution ofsingle-occupied partitions while providing lower levels of unoccupiedpartitions. The above noted ranges of unoccupied partitions can beachieved while still providing any of the single occupancy ratesdescribed above. For example, in many cases, the use of the systems andmethods described herein can create resulting partitions that havemultiple occupancy rates of less than about 25%, less than about 20%,less than about 15%, less than about 10%, and in many cases, less thanabout 5%, while having unoccupied partitions of less than about 50%,less than about 40%, less than about 30%, less than about 20%, less thanabout 10%, less than about 5%, or less.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both analyte carriers andadditional reagents, including, but not limited to, microcapsules orbeads (e.g., gel beads) carrying barcoded nucleic acid molecules (e.g.,oligonucleotides) (described in relation to FIG. 2). The occupiedpartitions (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, or 99% of the occupied partitions) can include both amicrocapsule (e.g., bead) comprising barcoded nucleic acid molecules andan analyte carrier.

In another aspect, in addition to or as an alternative to droplet basedpartitioning, analyte carriers may be encapsulated within a microcapsulethat comprises an outer shell, layer or porous matrix in which isentrained one or more individual analyte carriers or small groups ofanalyte carriers. The microcapsule may include other reagents.Encapsulation of analyte carriers may be performed by a variety ofprocesses. Such processes may combine an aqueous fluid containing theanalyte carriers with a polymeric precursor material that may be capableof being formed into a gel or other solid or semi-solid matrix uponapplication of a particular stimulus to the polymer precursor. Suchstimuli can include, for example, thermal stimuli (e.g., either heatingor cooling), photo-stimuli (e.g., through photo-curing), chemicalstimuli (e.g., through crosslinking, polymerization initiation of theprecursor (e.g., through added initiators)), mechanical stimuli, or acombination thereof.

Preparation of microcapsules comprising analyte carriers may beperformed by a variety of methods. For example, air knife droplet oraerosol generators may be used to dispense droplets of precursor fluidsinto gelling solutions in order to form microcapsules that includeindividual analyte carriers or small groups of analyte carriers.Likewise, membrane based encapsulation systems may be used to generatemicrocapsules comprising encapsulated analyte carriers as describedherein. Microfluidic systems of the present disclosure, such as thatshown in FIG. 1, may be readily used in encapsulating cells as describedherein. In particular, and with reference to FIG. 1, the aqueous fluid112 comprising (i) the analyte carriers 114 and (ii) the polymerprecursor material (not shown) is flowed into channel junction 110,where it is partitioned into droplets 118, 120 through the flow ofnon-aqueous fluid 116. In the case of encapsulation methods, non-aqueousfluid 116 may also include an initiator (not shown) to causepolymerization and/or crosslinking of the polymer precursor to form themicrocapsule that includes the entrained analyte carriers. Examples ofpolymer precursor/initiator pairs include those described in U.S. PatentApplication Publication No. 2014/0378345, which is entirely incorporatedherein by reference for all purposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, such as a linear polyacrylamide, PEG, orother linear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) comonomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams 116 in channel segments 104 and 106, which can initiatethe copolymerization of the acrylamide and BAC into a cross-linkedpolymer network, or hydrogel.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110, during formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous fluid 112 comprising thelinear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets 118, 120, resulting in the formationof gel (e.g., hydrogel) microcapsules, as solid or semi-solid beads orparticles entraining the cells 114. Although described in terms ofpolyacrylamide encapsulation, other ‘activatable’ encapsulationcompositions may also be employed in the context of the methods andcompositions described herein. For example, formation of alginatedroplets followed by exposure to divalent metal ions (e.g., Ca²⁺ ions),can be used as an encapsulation process using the described processes.Likewise, agarose droplets may also be transformed into capsules throughtemperature based gelling (e.g., upon cooling, etc.).

In some cases, encapsulated analyte carriers can be selectivelyreleasable from the microcapsule, such as through passage of time orupon application of a particular stimulus, that degrades themicrocapsule sufficiently to allow the analyte carriers (e.g., cell), orits other contents to be released from the microcapsule, such as into apartition (e.g., droplet). For example, in the case of thepolyacrylamide polymer described above, degradation of the microcapsulemay be accomplished through the introduction of an appropriate reducingagent, such as DTT or the like, to cleave disulfide bonds thatcross-link the polymer matrix. See, for example, U.S. Patent ApplicationPublication No. 2014/0378345, which is entirely incorporated herein byreference for all purposes.

The analyte carrier can be subjected to other conditions sufficient topolymerize or gel the precursors. The conditions sufficient topolymerize or gel the precursors may comprise exposure to heating,cooling, electromagnetic radiation, and/or light. The conditionssufficient to polymerize or gel the precursors may comprise anyconditions sufficient to polymerize or gel the precursors. Followingpolymerization or gelling, a polymer or gel may be formed around theanalyte carrier. The polymer or gel may be diffusively permeable tochemical or biochemical reagents. The polymer or gel may be diffusivelyimpermeable to macromolecular constituents of the analyte carrier. Inthis manner, the polymer or gel may act to allow the analyte carrier tobe subjected to chemical or biochemical operations while spatiallyconfining the macromolecular constituents to a region of the dropletdefined by the polymer or gel. The polymer or gel may include one ormore of disulfide cross-linked polyacrylamide, agarose, alginate,polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate,PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronicacid, collagen, fibrin, gelatin, or elastin. The polymer or gel maycomprise any other polymer or gel.

The polymer or gel may be functionalized to bind to targeted analytes,such as nucleic acids, proteins, carbohydrates, lipids or otheranalytes. The polymer or gel may be polymerized or gelled via a passivemechanism. The polymer or gel may be stable in alkaline conditions or atelevated temperature. The polymer or gel may have mechanical propertiessimilar to the mechanical properties of the bead. For instance, thepolymer or gel may be of a similar size to the bead. The polymer or gelmay have a mechanical strength (e.g. tensile strength) similar to thatof the bead. The polymer or gel may be of a lower density than an oil.The polymer or gel may be of a density that is roughly similar to thatof a buffer. The polymer or gel may have a tunable pore size. The poresize may be chosen to, for instance, retain denatured nucleic acids. Thepore size may be chosen to maintain diffusive permeability to exogenouschemicals such as sodium hydroxide (NaOH) and/or endogenous chemicalssuch as inhibitors. The polymer or gel may be biocompatible. The polymeror gel may maintain or enhance cell viability. The polymer or gel may bebiochemically compatible. The polymer or gel may be polymerized and/ordepolymerized thermally, chemically, enzymatically, and/or optically.

The polymer may comprise poly(acrylamide-co-acrylic acid) crosslinkedwith disulfide linkages. The preparation of the polymer may comprise atwo-step reaction. In the first activation step,poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent toconvert carboxylic acids to esters. For instance, thepoly(acrylamide-co-acrylic acid) may be exposed to4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM). The polyacrylamide-co-acrylic acid may be exposed to othersalts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. Inthe second cross-linking step, the ester formed in the first step may beexposed to a disulfide crosslinking agent. For instance, the ester maybe exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the twosteps, the analyte carrier may be surrounded by polyacrylamide strandslinked together by disulfide bridges. In this manner, the analytecarrier may be encased inside of or comprise a gel or matrix (e.g.,polymer matrix) to form a “cell bead.” A cell bead can contain analytecarriers (e.g., a cell) or macromolecular constituents (e.g., RNA, DNA,proteins, etc.) of analyte carriers. A cell bead may include a singlecell or multiple cells, or a derivative of the single cell or multiplecells. For example after lysing and washing the cells, inhibitorycomponents from cell lysates can be washed away and the macromolecularconstituents can be bound as cell beads. Systems and methods disclosedherein can be applicable to both cell beads (and/or droplets or otherpartitions) containing analyte carriers and cell beads (and/or dropletsor other partitions) containing macromolecular constituents of analytecarriers.

Encapsulated analyte carriers can provide certain potential advantagesof being more storable and more portable than droplet-based partitionedanalyte carriers. Furthermore, in some cases, it may be desirable toallow analyte carriers to incubate for a select period of time beforeanalysis, such as in order to characterize changes in such analytecarriers over time, either in the presence or absence of differentstimuli. In such cases, encapsulation may allow for longer incubationthan partitioning in emulsion droplets, although in some cases, dropletpartitioned analyte carriers may also be incubated for different periodsof time, e.g., at least 10 seconds, at least 30 seconds, at least 1minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, atleast 1 hour, at least 2 hours, at least 5 hours, or at least 10 hoursor more. The encapsulation of analyte carriers may constitute thepartitioning of the analyte carriers into which other reagents areco-partitioned. Alternatively or in addition, encapsulated analytecarriers may be readily deposited into other partitions (e.g., droplets)as described above.

Beads

A partition may comprise one or more unique identifiers, such asbarcodes. Barcodes may be previously, subsequently or concurrentlydelivered to the partitions that hold the compartmentalized orpartitioned analyte carrier. For example, barcodes may be injected intodroplets previous to, subsequent to, or concurrently with dropletgeneration. The delivery of the barcodes to a particular partitionallows for the later attribution of the characteristics of theindividual analyte carrier to the particular partition. Barcodes may bedelivered, for example on a nucleic acid molecule (e.g., anoligonucleotide), to a partition via any suitable mechanism. Barcodednucleic acid molecules can be delivered to a partition via amicrocapsule. A microcapsule, in some instances, can comprise a bead.Beads are described in further detail below.

In some cases, barcoded nucleic acid molecules can be initiallyassociated with the microcapsule and then released from themicrocapsule. Release of the barcoded nucleic acid molecules can bepassive (e.g., by diffusion out of the microcapsule). In addition oralternatively, release from the microcapsule can be upon application ofa stimulus which allows the barcoded nucleic acid nucleic acid moleculesto dissociate or to be released from the microcapsule. Such stimulus maydisrupt the microcapsule, an interaction that couples the barcodednucleic acid molecules to or within the microcapsule, or both. Suchstimulus can include, for example, a thermal stimulus, photo-stimulus,chemical stimulus (e.g., change in pH or use of a reducing agent(s)), amechanical stimulus, a radiation stimulus; a biological stimulus (e.g.,enzyme), or any combination thereof.

FIG. 2 shows an example of a microfluidic channel structure 200 fordelivering barcode carrying beads to droplets. The channel structure 200can include channel segments 201, 202, 204, 206 and 208 communicating ata channel junction 210. In operation, the channel segment 201 maytransport an aqueous fluid 212 that includes a plurality of beads 214(e.g., with nucleic acid molecules, oligonucleotides, molecular tags)along the channel segment 201 into junction 210. The plurality of beads214 may be sourced from a suspension of beads. For example, the channelsegment 201 may be connected to a reservoir comprising an aqueoussuspension of beads 214. The channel segment 202 may transport theaqueous fluid 212 that includes a plurality of analyte carriers 216along the channel segment 202 into junction 210. The plurality ofanalyte carriers 216 may be sourced from a suspension of analytecarriers. For example, the channel segment 202 may be connected to areservoir comprising an aqueous suspension of analyte carriers 216. Insome instances, the aqueous fluid 212 in either the first channelsegment 201 or the second channel segment 202, or in both segments, caninclude one or more reagents, as further described below. A second fluid218 that is immiscible with the aqueous fluid 212 (e.g., oil) can bedelivered to the junction 210 from each of channel segments 204 and 206.Upon meeting of the aqueous fluid 212 from each of channel segments 201and 202 and the second fluid 218 from each of channel segments 204 and206 at the channel junction 210, the aqueous fluid 212 can bepartitioned as discrete droplets 220 in the second fluid 218 and flowaway from the junction 210 along channel segment 208. The channelsegment 208 may deliver the discrete droplets to an outlet reservoirfluidly coupled to the channel segment 208, where they may be harvested.

As an alternative, the channel segments 201 and 202 may meet at anotherjunction upstream of the junction 210. At such junction, beads andanalyte carriers may form a mixture that is directed along anotherchannel to the junction 210 to yield droplets 220. The mixture mayprovide the beads and analyte carriers in an alternating fashion, suchthat, for example, a droplet comprises a single bead and a singleanalyte carrier.

Beads, analyte carriers and droplets may flow along channels atsubstantially regular flow profiles (e.g., at regular flow rates). Suchregular flow profiles may permit a droplet to include a single bead anda single analyte carrier. Such regular flow profiles may permit thedroplets to have an occupancy (e.g., droplets having beads and analytecarriers) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 95%. Such regular flow profiles and devices that may be used toprovide such regular flow profiles are provided in, for example, U.S.Patent Publication No. 2015/0292988, which is entirely incorporatedherein by reference.

The second fluid 218 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets220.

A discrete droplet that is generated may include an individual analytecarrier 216. A discrete droplet that is generated may include a barcodeor other reagent carrying bead 214. A discrete droplet generated mayinclude both an individual analyte carrier and a barcode carrying bead,such as droplets 220. In some instances, a discrete droplet may includemore than one individual analyte carrier or no analyte carrier. In someinstances, a discrete droplet may include more than one bead or no bead.A discrete droplet may be unoccupied (e.g., no beads, no analytecarriers).

Beneficially, a discrete droplet partitioning an analyte carrier and abarcode carrying bead may effectively allow the attribution of thebarcode to macromolecular constituents of the analyte carrier within thepartition. The contents of a partition may remain discrete from thecontents of other partitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 200 may have other geometries. For example, amicrofluidic channel structure can have more than one channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, or 5channel segments each carrying beads that meet at a channel junction.Fluid may be directed flow along one or more channels or reservoirs viaone or more fluid flow units. A fluid flow unit can comprise compressors(e.g., providing positive pressure), pumps (e.g., providing negativepressure), actuators, and the like to control flow of the fluid. Fluidmay also or otherwise be controlled via applied pressure differentials,centrifugal force, electrokinetic pumping, vacuum, capillary or gravityflow, or the like.

A bead may be porous, non-porous, solid, semi-solid, semi-fluidic,fluidic, and/or a combination thereof. In some instances, a bead may bedissolvable, disruptable, and/or degradable. In some cases, a bead maynot be degradable. In some cases, the bead may be a gel bead. A gel beadmay be a hydrogel bead. A gel bead may be formed from molecularprecursors, such as a polymeric or monomeric species. A semi-solid beadmay be a liposomal bead. Solid beads may comprise metals including ironoxide, gold, and silver. In some cases, the bead may be a silica bead.In some cases, the bead can be rigid. In other cases, the bead may beflexible and/or compressible.

A bead may be of any suitable shape. Examples of bead shapes include,but are not limited to, spherical, non-spherical, oval, oblong,amorphous, circular, cylindrical, and variations thereof.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be at least about 10 nanometers (nm), 100 nm, 500nm, 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm,70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm, or greater. In somecases, a bead may have a diameter of less than about 10 nm, 100 nm, 500nm, lμm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm,90 μm, 100 μm, 250 μm, 500 μm, 1 mm, or less. In some cases, a bead mayhave a diameter in the range of about 40-75 μm, 30-75 μm, 20-75 μm,40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500μm.

In certain aspects, beads can be provided as a population or pluralityof beads having a relatively monodisperse size distribution. Where itmay be desirable to provide relatively consistent amounts of reagentswithin partitions, maintaining relatively consistent beadcharacteristics, such as size, can contribute to the overallconsistency. In particular, the beads described herein may have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than 50%, less than 40%, less than30%, less than 20%, and in some cases less than 15%, less than 10%, lessthan 5%, or less.

A bead may comprise natural and/or synthetic materials. For example, abead can comprise a natural polymer, a synthetic polymer or both naturaland synthetic polymers. Examples of natural polymers include proteinsand sugars such as deoxyribonucleic acid, rubber, cellulose, starch(e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silks,polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan,ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum,Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate,or natural polymers thereof. Examples of synthetic polymers includeacrylics, nylons, silicones, spandex, viscose rayon, polycarboxylicacids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethyleneglycol, polyurethanes, polylactic acid, silica, polystyrene,polyacrylonitrile, polybutadiene, polycarbonate, polyethylene,polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethyleneoxide), poly(ethylene terephthalate), polyethylene, polyisobutylene,poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde,polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinylacetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidenedichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/orcombinations (e.g., co-polymers) thereof. Beads may also be formed frommaterials other than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some instances, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the molecular precursors. In some cases, a precursormay be an already polymerized species capable of undergoing furtherpolymerization via, for example, a chemical cross-linkage. In somecases, a precursor can comprise one or more of an acrylamide or amethacrylamide monomer, oligomer, or polymer. In some cases, the beadmay comprise prepolymers, which are oligomers capable of furtherpolymerization. For example, polyurethane beads may be prepared usingprepolymers. In some cases, the bead may contain individual polymersthat may be further polymerized together. In some cases, beads may begenerated via polymerization of different precursors, such that theycomprise mixed polymers, co-polymers, and/or block co-polymers. In somecases, the bead may comprise covalent or ionic bonds between polymericprecursors (e.g., monomers, oligomers, linear polymers), nucleic acidmolecules (e.g., oligonucleotides), primers, and other entities. In somecases, the covalent bonds can be carbon-carbon bonds, thioether bonds,or carbon-heteroatom bonds.

Cross-linking may be permanent or reversible, depending upon theparticular cross-linker used. Reversible cross-linking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some cases, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and nucleic acid molecules (e.g.,oligonucleotides). Cystamine (including modified cystamines), forexample, is an organic agent comprising a disulfide bond that may beused as a crosslinker agent between individual monomeric or polymericprecursors of a bead. Polyacrylamide may be polymerized in the presenceof cystamine or a species comprising cystamine (e.g., a modifiedcystamine) to generate polyacrylamide gel beads comprising disulfidelinkages (e.g., chemically degradable beads comprisingchemically-reducible cross-linkers). The disulfide linkages may permitthe bead to be degraded (or dissolved) upon exposure of the bead to areducing agent.

In some cases, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more nucleic acid molecules (e.g.,barcode sequence, barcoded nucleic acid molecule, barcodedoligonucleotide, primer, or other oligonucleotide) to the bead. In somecases, an acrydite moiety can refer to an acrydite analogue generatedfrom the reaction of acrydite with one or more species, such as, thereaction of acrydite with other monomers and cross-linkers during apolymerization reaction. Acrydite moieties may be modified to formchemical bonds with a species to be attached, such as a nucleic acidmolecule (e.g., barcode sequence, barcoded nucleic acid molecule,barcoded oligonucleotide, primer, or other oligonucleotide). Acryditemoieties may be modified with thiol groups capable of forming adisulfide bond or may be modified with groups already comprising adisulfide bond. The thiol or disulfide (via disulfide exchange) may beused as an anchor point for a species to be attached or another part ofthe acrydite moiety may be used for attachment. In some cases,attachment can be reversible, such that when the disulfide bond isbroken (e.g., in the presence of a reducing agent), the attached speciesis released from the bead. In other cases, an acrydite moiety cancomprise a reactive hydroxyl group that may be used for attachment.

Functionalization of beads for attachment of nucleic acid molecules(e.g., oligonucleotides) may be achieved through a wide range ofdifferent approaches, including activation of chemical groups within apolymer, incorporation of active or activatable functional groups in thepolymer structure, or attachment at the pre-polymer or monomer stage inbead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to a nucleic acid molecule (e.g.,oligonucleotide), which may include a priming sequence (e.g., a primerfor amplifying target nucleic acids, random primer, primer sequence formessenger RNA) and/or one or more barcode sequences. The one morebarcode sequences may include sequences that are the same for allnucleic acid molecules coupled to a given bead and/or sequences that aredifferent across all nucleic acid molecules coupled to the given bead.The nucleic acid molecule may be incorporated into the bead. In somecases, the nucleic acid molecule can comprise a functional sequence, forexample, for attachment to a sequencing flow cell, such as, for example,a P5 sequence for Illumina® sequencing. In some cases, the nucleic acidmolecule or derivative thereof (e.g., oligonucleotide or polynucleotidegenerated from the nucleic acid molecule) can comprise anotherfunctional sequence, such as, for example, a P7 sequence for attachmentto a sequencing flow cell for Illumina sequencing. In some cases, thenucleic acid molecule can comprise a barcode sequence. In some cases,the primer can further comprise a unique molecular identifier (UMI). Insome cases, the primer can comprise an R1 primer sequence for Illuminasequencing. In some cases, the primer can comprise an R2 primer sequencefor Illumina sequencing. Examples of such nucleic acid molecules (e.g.,oligonucleotides, polynucleotides, etc.) and uses thereof, as may beused with compositions, devices, methods and systems of the presentdisclosure, are provided in U.S. Patent Pub. Nos. 2014/0378345 and2015/0376609, each of which is entirely incorporated herein byreference.

FIG. 8 illustrates an example of a barcode carrying bead. A nucleic acidmolecule 802, such as an oligonucleotide, can be coupled to a bead 804by a releasable linkage 806, such as, for example, a disulfide linker.The same bead 804 may be coupled (e.g., via releasable linkage) to oneor more other nucleic acid molecules 818, 820. The nucleic acid molecule802 may be or comprise a barcode. As noted elsewhere herein, thestructure of the barcode may comprise a number of sequence elements. Thenucleic acid molecule 802 may comprise a functional sequence 808 thatmay be used in subsequent processing. For example, the functionalsequence 808 may include one or more of a sequencer specific flow cellattachment sequence (e.g., a P5 sequence for Illumina® sequencingsystems) and a sequencing primer sequence (e.g., a R1 primer forIllumina® sequencing systems). The nucleic acid molecule 802 maycomprise a barcode sequence 810 for use in barcoding the sample (e.g.,DNA, RNA, protein, etc.). In some cases, the barcode sequence 810 can bebead-specific such that the barcode sequence 810 is common to allnucleic acid molecules (e.g., including nucleic acid molecule 802)coupled to the same bead 804. Alternatively or in addition, the barcodesequence 810 can be partition-specific such that the barcode sequence810 is common to all nucleic acid molecules coupled to one or more beadsthat are partitioned into the same partition. The nucleic acid molecule802 may comprise a specific priming sequence 812, such as an mRNAspecific priming sequence (e.g., poly-T sequence), a targeted primingsequence, and/or a random priming sequence. The nucleic acid molecule802 may comprise an anchoring sequence 814 to ensure that the specificpriming sequence 812 hybridizes at the sequence end (e.g., of the mRNA).For example, the anchoring sequence 814 can include a random shortsequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longersequence, which can ensure that a poly-T segment is more likely tohybridize at the sequence end of the poly-A tail of the mRNA.

The nucleic acid molecule 802 may comprise a unique molecularidentifying sequence 816 (e.g., unique molecular identifier (UMI)). Insome cases, the unique molecular identifying sequence 816 may comprisefrom about 5 to about 8 nucleotides. Alternatively, the unique molecularidentifying sequence 816 may compress less than about 5 or more thanabout 8 nucleotides. The unique molecular identifying sequence 816 maybe a unique sequence that varies across individual nucleic acidmolecules (e.g., 802, 818, 820, etc.) coupled to a single bead (e.g.,bead 804). In some cases, the unique molecular identifying sequence 816may be a random sequence (e.g., such as a random N-mer sequence). Forexample, the UMI may provide a unique identifier of the starting mRNAmolecule that was captured, in order to allow quantification of thenumber of original expressed RNA. As will be appreciated, although FIG.8 shows three nucleic acid molecules 802, 818, 820 coupled to thesurface of the bead 804, an individual bead may be coupled to any numberof individual nucleic acid molecules, for example, from one to tens tohundreds of thousands or even millions of individual nucleic acidmolecules. The respective barcodes for the individual nucleic acidmolecules can comprise both common sequence segments or relativelycommon sequence segments (e.g., 808, 810, 812, etc.) and variable orunique sequence segments (e.g., 816) between different individualnucleic acid molecules coupled to the same bead.

In operation, an analyte carrier (e.g., cell, DNA, RNA, etc.) can beco-partitioned along with a barcode bearing bead 804. The barcodednucleic acid molecules 802, 818, 820 can be released from the bead 804in the partition. By way of example, in the context of analyzing sampleRNA, the poly-T segment (e.g., 812) of one of the released nucleic acidmolecules (e.g., 802) can hybridize to the poly-A tail of an mRNAmolecule. Reverse transcription may result in a cDNA transcript of themRNA, but which transcript includes each of the sequence segments 808,810, 816 of the nucleic acid molecule 802. Because the nucleic acidmolecule 802 comprises an anchoring sequence 814, it will more likelyhybridize to and prime reverse transcription at the sequence end of thepoly-A tail of the mRNA. Within any given partition, all of the cDNAtranscripts of the individual mRNA molecules may include a commonbarcode sequence segment 810. However, the transcripts made from thedifferent mRNA molecules within a given partition may vary at the uniquemolecular identifying sequence 812 segment (e.g., UMI segment).Beneficially, even following any subsequent amplification of thecontents of a given partition, the number of different UMIs can beindicative of the quantity of mRNA originating from a given partition,and thus from the analyte carrier (e.g., cell). As noted above, thetranscripts can be amplified, cleaned up and sequenced to identify thesequence of the cDNA transcript of the mRNA, as well as to sequence thebarcode segment and the UMI segment. While a poly-T primer sequence isdescribed, other targeted or random priming sequences may also be usedin priming the reverse transcription reaction. Likewise, althoughdescribed as releasing the barcoded oligonucleotides into the partition,in some cases, the nucleic acid molecules bound to the bead (e.g., gelbead) may be used to hybridize and capture the mRNA on the solid phaseof the bead, for example, in order to facilitate the separation of theRNA from other cell contents.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can co-polymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group. In some cases, acrylic acid (a species comprising freeCOOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a gel bead comprising free COOHgroups. The COOH groups of the gel bead can be activated (e.g., via1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NETS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than or equal to about 1:100,000,000,000, less than orequal to about 1:10,000,000,000, less than or equal to about1:1,000,000,000, less than or equal to about 1:100,000,000, less than orequal to about 1:10,000,000, less than or equal to about 1:1,000,000,less than or equal to about 1:100,000, less than or equal to about1:10,000) of reducing agent may be used for reduction. Controlling thenumber of disulfide linkages that are reduced to free thiols may beuseful in ensuring bead structural integrity during functionalization.In some cases, optically-active agents, such as fluorescent dyes may becoupled to beads via free thiol groups of the beads and used to quantifythe number of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of anoligonucleotide (e.g., barcoded oligonucleotide) after gel beadformation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading may also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

A bead injected or otherwise introduced into a partition may comprisereleasably, cleavably, or reversibly attached barcodes. A bead injectedor otherwise introduced into a partition may comprise activatablebarcodes. A bead injected or otherwise introduced into a partition maybe degradable, disruptable, or dissolvable beads.

Barcodes can be releasably, cleavably or reversibly attached to thebeads such that barcodes can be released or be releasable throughcleavage of a linkage between the barcode molecule and the bead, orreleased through degradation of the underlying bead itself, allowing thebarcodes to be accessed or be accessible by other reagents, or both. Innon-limiting examples, cleavage may be achieved through reduction ofdi-sulfide bonds, use of restriction enzymes, photo-activated cleavage,or cleavage via other types of stimuli (e.g., chemical, thermal, pH,enzymatic, etc.) and/or reactions, such as described elsewhere herein.Releasable barcodes may sometimes be referred to as being activatable,in that they are available for reaction once released. Thus, forexample, an activatable barcode may be activated by releasing thebarcode from a bead (or other suitable type of partition describedherein). Other activatable configurations are also envisioned in thecontext of the described methods and systems.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, such as barcode containingnucleic acid molecules (e.g., barcoded oligonucleotides), the beads maybe degradable, disruptable, or dissolvable spontaneously or uponexposure to one or more stimuli (e.g., temperature changes, pH changes,exposure to particular chemical species or phase, exposure to light,reducing agent, etc.). In some cases, a bead may be dissolvable, suchthat material components of the beads are solubilized when exposed to aparticular chemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead can be degradedor dissolved at elevated temperature and/or in basic conditions. In somecases, a bead may be thermally degradable such that when the bead isexposed to an appropriate change in temperature (e.g., heat), the beaddegrades. Degradation or dissolution of a bead bound to a species (e.g.,a nucleic acid molecule, e.g., barcoded oligonucleotide) may result inrelease of the species from the bead.

As will be appreciated from the above disclosure, the degradation of abead may refer to the disassociation of a bound or entrained speciesfrom a bead, both with and without structurally degrading the physicalbead itself. For example, the degradation of the bead may involvecleavage of a cleavable linkage via one or more species and/or methodsdescribed elsewhere herein. In another example, entrained species may bereleased from beads through osmotic pressure differences due to, forexample, changing chemical environments. By way of example, alterationof bead pore sizes due to osmotic pressure differences can generallyoccur without structural degradation of the bead itself. In some cases,an increase in pore size due to osmotic swelling of a bead can permitthe release of entrained species within the bead. In other cases,osmotic shrinking of a bead may cause a bead to better retain anentrained species due to pore size contraction.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides) arereleased within the droplet when the appropriate stimulus is applied.The free species (e.g., oligonucleotides, nucleic acid molecules) mayinteract with other reagents contained in the partition. For example, apolyacrylamide bead comprising cystamine and linked, via a disulfidebond, to a barcode sequence, may be combined with a reducing agentwithin a droplet of a water-in-oil emulsion. Within the droplet, thereducing agent can break the various disulfide bonds, resulting in beaddegradation and release of the barcode sequence into the aqueous, innerenvironment of the droplet. In another example, heating of a dropletcomprising a bead-bound barcode sequence in basic solution may alsoresult in bead degradation and release of the attached barcode sequenceinto the aqueous, inner environment of the droplet.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingnucleic acid molecule (e.g., oligonucleotide) bearing beads.

In some cases, beads can be non-covalently loaded with one or morereagents. The beads can be non-covalently loaded by, for instance,subjecting the beads to conditions sufficient to swell the beads,allowing sufficient time for the reagents to diffuse into the interiorsof the beads, and subjecting the beads to conditions sufficient tode-swell the beads. The swelling of the beads may be accomplished, forinstance, by placing the beads in a thermodynamically favorable solvent,subjecting the beads to a higher or lower temperature, subjecting thebeads to a higher or lower ion concentration, and/or subjecting thebeads to an electric field. The swelling of the beads may beaccomplished by various swelling methods. The de-swelling of the beadsmay be accomplished, for instance, by transferring the beads in athermodynamically unfavorable solvent, subjecting the beads to lower orhigh temperatures, subjecting the beads to a lower or higher ionconcentration, and/or removing an electric field. The de-swelling of thebeads may be accomplished by various de-swelling methods. Transferringthe beads may cause pores in the bead to shrink. The shrinking may thenhinder reagents within the beads from diffusing out of the interiors ofthe beads. The hindrance may be due to steric interactions between thereagents and the interiors of the beads. The transfer may beaccomplished microfluidically. For instance, the transfer may beachieved by moving the beads from one co-flowing solvent stream to adifferent co-flowing solvent stream. The swellability and/or pore sizeof the beads may be adjusted by changing the polymer composition of thebead.

In some cases, an acrydite moiety linked to a precursor, another specieslinked to a precursor, or a precursor itself can comprise a labile bond,such as chemically, thermally, or photo-sensitive bond e.g., disulfidebond, UV sensitive bond, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, etc.) to a bead. In some cases, a thermally labilebond may include a nucleic acid hybridization based attachment, e.g.,where an oligonucleotide is hybridized to a complementary sequence thatis attached to the bead, such that thermal melting of the hybridreleases the oligonucleotide, e.g., a barcode containing sequence, fromthe bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead may resultin the generation of a bead capable of responding to varied stimuli.Each type of labile bond may be sensitive to an associated stimulus(e.g., chemical stimulus, light, temperature, enzymatic, etc.) such thatrelease of species attached to a bead via each labile bond may becontrolled by the application of the appropriate stimulus. Suchfunctionality may be useful in controlled release of species from a gelbead. In some cases, another species comprising a labile bond may belinked to a gel bead after gel bead formation via, for example, anactivated functional group of the gel bead as described above. As willbe appreciated, barcodes that are releasably, cleavably or reversiblyattached to the beads described herein include barcodes that arereleased or releasable through cleavage of a linkage between the barcodemolecule and the bead, or that are released through degradation of theunderlying bead itself, allowing the barcodes to be accessed oraccessible by other reagents, or both.

The barcodes that are releasable as described herein may sometimes bereferred to as being activatable, in that they are available forreaction once released. Thus, for example, an activatable barcode may beactivated by releasing the barcode from a bead (or other suitable typeof partition described herein). Other activatable configurations arealso envisioned in the context of the described methods and systems.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNAase)). A bond may be cleavable via other nucleic acid moleculetargeting enzymes, such as restriction enzymes (e.g., restrictionendonucleases), as described further below.

Species may be encapsulated in beads during bead generation (e.g.,during polymerization of precursors). Such species may or may notparticipate in polymerization. Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,nucleic acid molecules (e.g., oligonucleotides), reagents for a nucleicacid amplification reaction (e.g., primers, polymerases, dNTPs,co-factors (e.g., ionic co-factors), buffers) including those describedherein, reagents for enzymatic reactions (e.g., enzymes, co-factors,substrates, buffers), reagents for nucleic acid modification reactionssuch as polymerization, ligation, or digestion, and/or reagents fortemplate preparation (e.g., tagmentation) for one or more sequencingplatforms (e.g., Nextera® for Illumina®). Such species may include oneor more enzymes described herein, including without limitation,polymerase, reverse transcriptase, restriction enzymes (e.g.,endonuclease), transposase, ligase, proteinase K, DNAse, etc. Suchspecies may include one or more reagents described elsewhere herein(e.g., lysis agents, inhibitors, inactivating agents, chelating agents,stimulus). Trapping of such species may be controlled by the polymernetwork density generated during polymerization of precursors, controlof ionic charge within the gel bead (e.g., via ionic species linked topolymerized species), or by the release of other species. Encapsulatedspecies may be released from a bead upon bead degradation and/or byapplication of a stimulus capable of releasing the species from thebead. Alternatively or in addition, species may be partitioned in apartition (e.g., droplet) during or subsequent to partition formation.Such species may include, without limitation, the abovementioned speciesthat may also be encapsulated in a bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., a nucleic acid molecule, a barcode sequence, a primer,etc) from the bead when the appropriate stimulus is applied to the beadas compared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence may be attached, via a disulfide bond, to a polyacrylamide beadcomprising cystamine. Upon exposure of the barcoded-bead to a reducingagent, the bead degrades and the barcode sequence is released uponbreakage of both the disulfide linkage between the barcode sequence andthe bead and the disulfide linkages of the cystamine in the bead.

As will be appreciated from the above disclosure, while referred to asdegradation of a bead, in many instances as noted above, thatdegradation may refer to the disassociation of a bound or entrainedspecies from a bead, both with and without structurally degrading thephysical bead itself. For example, entrained species may be releasedfrom beads through osmotic pressure differences due to, for example,changing chemical environments. By way of example, alteration of beadpore sizes due to osmotic pressure differences can generally occurwithout structural degradation of the bead itself. In some cases, anincrease in pore size due to osmotic swelling of a bead can permit therelease of entrained species within the bead. In other cases, osmoticshrinking of a bead may cause a bead to better retain an entrainedspecies due to pore size contraction.

Where degradable beads are provided, it may be beneficial to avoidexposing such beads to the stimulus or stimuli that cause suchdegradation prior to a given time, in order to, for example, avoidpremature bead degradation and issues that arise from such degradation,including for example poor flow characteristics and aggregation. By wayof example, where beads comprise reducible cross-linking groups, such asdisulfide groups, it will be desirable to avoid contacting such beadswith reducing agents, e.g., DTT or other disulfide cleaving reagents. Insuch cases, treatment to the beads described herein will, in some casesbe provided free of reducing agents, such as DTT. Because reducingagents are often provided in commercial enzyme preparations, it may bedesirable to provide reducing agent free (or DTT free) enzymepreparations in treating the beads described herein. Examples of suchenzymes include, e.g., polymerase enzyme preparations, reversetranscriptase enzyme preparations, ligase enzyme preparations, as wellas many other enzyme preparations that may be used to treat the beadsdescribed herein. The terms “reducing agent free” or “DTT free”preparations can refer to a preparation having less than about 1/10th,less than about 1/50th, or even less than about 1/100th of the lowerranges for such materials used in degrading the beads. For example, forDTT, the reducing agent free preparation can have less than about 0.01millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even lessthan about 0.0001 mM DTT. In many cases, the amount of DTT can beundetectable.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead. In some cases, any combination of stimulimay trigger degradation of a bead. For example, a change in pH mayenable a chemical agent (e.g., DTT) to become an effective reducingagent.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

Any suitable agent may degrade beads. In some embodiments, changes intemperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM. The reducing agent may be present ata concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, orgreater than 10 mM. The reducing agent may be present at concentrationof at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

Any suitable number of molecular tag molecules (e.g., primer, barcodedoligonucleotide) can be associated with a bead such that, upon releasefrom the bead, the molecular tag molecules (e.g., primer, e.g., barcodedoligonucleotide) are present in the partition at a pre-definedconcentration. Such pre-defined concentration may be selected tofacilitate certain reactions for generating a sequencing library, e.g.,amplification, within the partition. In some cases, the pre-definedconcentration of the primer can be limited by the process of producingoligonucleotide bearing beads.

Although FIG. 1 and FIG. 2 have been described in terms of providingsubstantially singly occupied partitions, above, in certain cases, itmay be desirable to provide multiply occupied partitions, e.g.,containing two, three, four or more cells and/or microcapsules (e.g.,beads) comprising barcoded nucleic acid molecules (e.g.,oligonucleotides) within a single partition. Accordingly, as notedabove, the flow characteristics of the analyte carrier and/or beadcontaining fluids and partitioning fluids may be controlled to providefor such multiply occupied partitions. In particular, the flowparameters may be controlled to provide a given occupancy rate atgreater than about 50% of the partitions, greater than about 75%, and insome cases greater than about 80%, 90%, 95%, or higher.

In some cases, additional microcapsules can be used to deliveradditional reagents to a partition. In such cases, it may beadvantageous to introduce different beads into a common channel ordroplet generation junction, from different bead sources (e.g.,containing different associated reagents) through different channelinlets into such common channel or droplet generation junction (e.g.,junction 210). In such cases, the flow and frequency of the differentbeads into the channel or junction may be controlled to provide for acertain ratio of microcapsules from each source, while ensuring a givenpairing or combination of such beads into a partition with a givennumber of analyte carriers (e.g., one analyte carrier and one bead perpartition).

The partitions described herein may comprise small volumes, for example,less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL),800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL,20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than about 1000 pL, 900 pL, 800 pL,700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10pL, 1 pL, or less. Where co-partitioned with microcapsules, it will beappreciated that the sample fluid volume, e.g., including co-partitionedanalyte carriers and/or beads, within the partitions may be less thanabout 90% of the above described volumes, less than about 80%, less thanabout 70%, less than about 60%, less than about 50%, less than about40%, less than about 30%, less than about 20%, or less than about 10% ofthe above described volumes.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated or otherwise provided. Forexample, at least about 1,000 partitions, at least about 5,000partitions, at least about 10,000 partitions, at least about 50,000partitions, at least about 100,000 partitions, at least about 500,000partitions, at least about 1,000,000 partitions, at least about5,000,000 partitions at least about 10,000,000 partitions, at leastabout 50,000,000 partitions, at least about 100,000,000 partitions, atleast about 500,000,000 partitions, at least about 1,000,000,000partitions, or more partitions can be generated or otherwise provided.Moreover, the plurality of partitions may comprise both unoccupiedpartitions (e.g., empty partitions) and occupied partitions.

The compositions, methods and kits described herein can comprise aplurality of partitions, wherein one or more partitions of the pluralityof partitions may comprise one or more barcode-containing beads, one ormore synthetic cells (or control beads), or any combination thereof. Asdescribed herein, control beads can be used as control elements orinternal standards in the processing of a biological sample (e.g.,single cell analysis). A partition may comprise one or more differentbeads. Such a bead may be a barcode-containing bead or a control beadsuch as a synthetic cell. Such a bead as may be a control bead (e.g., asynthetic cell).

A bead may comprise various molecules. Such molecules may be nucleicacid molecules or polypeptides. A bead may be a control bead. A controlbead may be coupled to, attached to, or otherwise associated with amolecule using conjugation chemistry. Conjugation chemistry as describedherein refers to any chemical reaction that links, couples, or attachesa first molecule with a second molecule. Conjugation chemistry maycomprise bioconjugation chemistry and click chemistry. Conjugationchemistry may comprise biological interactions (e.g.,biotin/strepdavidin interactions) and/or bioorthogonal reactions.

In addition to using click chemistry, a molecule (e.g., a nucleic acidmolecule) can be attached to a bead (e.g., a gel bead) using variousother bioconjugation or coupling methods. Such bioconjugation methodscan include various conjugation strategies and functional groupmodifications such as mesylate formation, sulfur alkylation, NHS esterformation, carbamate formation, carbonate formation, amide bondformation, or any combination thereof. Such strategies and functionalgroup modifications can be used for various reaction types suchnucleophilic and/or electrophilic substitution reaction, nucleophilicand/or electrophilic addition reaction, and other suitable reactiontypes. In some cases, activated carboxylic acids can react withnucleophiles such as amines. In some cases, the carboxylic acid can beattached to a bead (e.g., a gel bead, control bead, etc.) and thenucleophilic group such as an amine can be attached to a molecule (e.g.,a nucleic acid molecule) to be attached to said bead. Such amide bondformation reactions can include EDC/NHS (e.g., via1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) andN-hydroxysuccinimide (NETS) or4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) mediated coupling reactions, wherein an activated ester (e.g.,an NHS ester attached to a bead surface) can react with an amine (e.g.,an amine of a nucleic acid molecule) to form an amide bond, therebyattaching said molecule (e.g., a nucleic acid molecule) to said bead(e.g., a gel bead). Any other suitable bioconjugation reactions can beused to attach a molecule to a bead.

Coupling or attachment of nucleic acid molecules to barcode beads and/orcontrol beads (e.g., synthetic cells) may be performed using clickchemistry. Click chemistry may comprise any type of click reactionsuitable for the functionalization of synthetic cells. Examples of clickchemistry reactions (or short “click reactions”) that may be used incombination with the herein described methods and compositions include,but are not limited to, transition-metal catalyzed or strain-promotedazide-alkyne cycloadditions (e.g., Huisgen azide-alkyne 1,3-dipolarcycloaddition, copper-catalyzed azide-alkyne cycloaddition (CuAAC),strain-promoted alkyne-azide cycloaddition, and/or ruthenium-catalyzedazide-alkyne cycloaddition (RuAAC)), Diels-Alder reactions such asinverse-electron demand Diels-Alder reaction (e.g.,tetrazine-trans-cyclooctene reactions), or photo-click reactions (e.g.,alkene-tetrazole photoreactions).

A bead may be attached to one or more sets of nucleic acid moleculesusing such click chemistry. Such a bead may be a control bead. A controlbead may comprise (e.g., may be functionalized with) a first functionalgroup. The first functional group may be a first reactant for a clickreaction. The one or more sets of nucleic acid molecules that may beattached to a control bead may comprise a second functional group. Thefirst functional group may be a second reactant for a click reaction.The click reaction is a copper-catalyzed azide-alkyne cycloadditionreaction, an inverse-electron demand Diels-Alder reaction, or anavidin-biotin interaction. In some instances, click reaction is acopper-catalyzed azide-alkyne cycloaddition reaction comprising anazide-functionalized nucleic acid molecule (e.g., a nucleic acidmolecule comprising a known sequence) and an alkyne-functionalized bead(e.g., a control bead).

The first functional group may react with a second functional group in aclick reaction. In some instances, a nucleic acid molecule comprising aknown sequence may be attached to a control bead by reacting said firstfunctional group with said second functional group. The first functionalgroup is an alkyne, a trans-cyclooctene, or an avidin, or anycombination thereof. In various instances, the first functional group isan alkyne. The second functional group is an azide, a tetrazine, or abiotin, or a combination thereof. In various instances, the secondfunctional group is an azide.

A nucleic acid molecule may be releasably attached to a bead (e.g., acontrol bead). A nucleic acid molecule may be releasably attached to abead via a releasable linkage. A nucleic acid molecule may be releasablyattached to a bead via a cleavable sequence. Such a cleavable sequencemay be a nucleotide sequence and/or an amino acid sequence. A cleavablesequence may release a nucleic acid molecule upon application of aparticular stimulus. Such stimuli can include, for example, thermalstimuli (e.g., either heating or cooling), photo-stimuli (e.g., throughphoto-curing), chemical stimuli (e.g., through crosslinking,polymerization initiation of the precursor (e.g., through addedinitiators), pH-mediated, etc.), mechanical stimuli, or a combinationthereof.

A nucleic acid molecule (or any other analyte) may be attached to a beadusing click reactions as described herein. Such click reactions maycomprise various reaction parameters. Such reaction parameters mayinclude reaction temperature, reaction time, and any additional reagentspresent in the reaction solution. The reaction parameters may depend onthe type of click reaction to be conducted. For example, acopper-catalyzed azide-alkyne cycloaddition reaction may comprise one ormore different reaction parameters than an avidin-biotin interaction.

A bead may be functionalized to comprise a first functional groupproviding reactivity for certain reactions (e.g., a click reaction witha functionalized nucleic acid molecule). A nucleic acid molecule to beattached to a bead may be functionalized to comprise a second functionalgroup providing reactivity for certain reactions (e.g., a click reactionwith a functionalized bead). A nucleic acid molecule may befunctionalized to comprise an azide moiety.

In operation, as illustrated in step 1102 of FIG. 11 (see also EXAMPLE1), a ligation reaction for modifying a nucleic acid molecule tocomprise an azide functional groups can comprise a solution (having aconcentration of e.g., 1-100 μM, 10-50 μM, or 5-20 μM) of synthesizednucleic acid (e.g., RNA), splint or bridge oligonucleotides (e.g., in aconcentration of 1-100 μM, 10-50 μM, or 5-20 μM), an azideoligonucleotide (e.g., in a concentration of 1-100 μM, 10-50 μM, or 5-20μM), and ligase, wherein the ligase may be present in a concentration ofabout 10-100 μg/μL, 1-100 μM, 10-50 μM, or 5-20 μM. The reactionsolution may be further diluted and/or adjusted to a specific volume byadding ligation buffer. As an example, a ligation reaction for modifyinga RNA molecule to comprise an azide functional groups can comprise 10 uMsynthesized RNA, 10 uM splint, 40 uM azide oligo, and 100 U/uL or 50ug/uL ligase, ligation buffer can be added up to a certain volume (e.g.,250 uL). The reaction may be incubated at a certain temperature (e.g.,about 16 degrees (° C.) for a certain period of time (e.g., 1-3 hours).Addition of EDTA (e.g., 1-10 μL or an amount to obtain a certainconcentration) may be added to the reaction solution to stop (or quench)the reaction. Quantitation of the ligation reaction may be performedusing various methods including the use of fluorescent dyes (e.g.,AlexaFluor488, AlexaFluor647 etc.) and/or instruments such as Nanodrop,etc. that allow high-throughput screening of various reactionconditions.

A bead comprising a first functional group may be attached to a nucleicacid molecule comprising a second functional group. The bead may beattached to the nucleic acid molecule via bioconjugation chemistry.Bioconjugation methods can include various conjugation strategies andfunctional group modifications such as mesylate formation, sulfuralkylation, NHS ester formation, carbamate formation, carbonateformation, amide bond formation, or any combination thereof. Suchstrategies and functional group modifications can be used for variousreaction types such nucleophilic and/or electrophilic substitutionreaction, nucleophilic and/or electrophilic addition reaction, and othersuitable reaction types. In some cases, activated carboxylic acids canreact with nucleophiles such as amines. In some cases, the carboxylicacid can be attached to a bead (e.g., a gel bead, control bead, etc.)and the nucleophilic group such as an amine can be attached to amolecule (e.g., a nucleic acid molecule) to be attached to said bead.Such amide bond formation reactions can include EDC/NHS (e.g., via1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) andN-hydroxysuccinimide (NETS) or4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) mediated coupling reactions, wherein an activated ester (e.g.,an NETS ester attached to a bead surface) can react with an amine (e.g.,an amine of a nucleic acid molecule) to form an amide bond, therebyattaching said molecule (e.g., a nucleic acid molecule) to said bead(e.g., a gel bead). Any other suitable bioconjugation reactions can beused to attach a molecule to a bead.

A nucleic acid molecule, or a modified derivative thereof, can beattached to a bead (e.g., a gel bead) using click chemistry. Inoperation, as illustrated in step 1301 of FIG. 13 (see also EXAMPLE 2),a nucleic acid molecule modified to comprise an azide moiety may beattached to a control bead via click chemistry. Such reactions mayinvolve one or more additional reagents and/or buffer solutions. Suchadditional reagents may include catalysts. Such a catalyst may compriseone or more transition metals (e.g., copper) and one or more ligands(e.g., THTPA). The click reaction between an azide-functionalizednucleic acid molecule and an alkyne-functionalized control bead may beperformed in a final reaction solution is which the click reaction isperformed may have a certain volume and certain final concentrations ofreactions. For example, a final reaction mixture for a copper-catalyzedazide-alkyne click reaction can comprise between about 100-1000 μL(e.g., 300 μL) of final volume, between about 0.5-5 mM (e.g., about 1.25mM) THPTA ligand, between about 0.1-0.75 (e.g., about 0.25 mM) CuAcO₄,between about 1-10 mM (e.g., about 5.0 mM) sodium ascorbate and betweenabout 0.01-0.1 mM (e.g., 0.04 mM) ligated (e.g., azide-functionalized)RNA, and between about 1-50 μM (e.g., 20 uM) gel beads such as controlbeads. As an example, a modified nucleic acid molecule comprising anazide moiety may be attached to an alkyne-functionalized control beadusing the ligand THPTA at 50 mM, 10 mM copper acetate, 100 mM sodiumascorbate, 0.16 mM ligated RNA, and 20 uM gel beads/synthetic cells.Such a click reaction may be performed at room temperature. Reactionsmay be stopped or quenched using EDTA, for example.

Any bead described herein may be attached to a nucleic acid moleculeusing conjugation chemistry (e.g., click chemistry). The bead to befunctionalized may be a control bead (e.g., synthetic cell) as describedherein, and as such, may be used as a quality control element in variouscell analyses processes, e.g., single-cell analyses of biologicalsamples. A control bead may comprise (e.g., be functionalized with) oneor more molecules. Such molecules may be the same molecules or may bedifferent molecules. The molecule that a control bead may befunctionalized with may be a nucleic acid molecule (e.g., RNA such asmRNA). Such a nucleic acid molecule may comprise a known sequence. Acontrol bead may comprise hundreds, thousands, or millions of nucleicacid molecules. Such nucleic acid molecules may have an identicalnucleic acid sequence or a different nucleic acid sequence. A controlbead may comprise at least two different nucleic acid molecules. Acontrol bead may comprise at least five different nucleic acidmolecules. A control bead may comprise at least ten different nucleicacid molecules. A control bead may comprise at least twenty differentnucleic acid molecules. A control bead may be functionalized with atleast fifty different nucleic acid molecules. A control bead may befunctionalized with at least a hundred different nucleic acid molecules.A control bead may be functionalized with about 1 to about 10 differentnucleic acid molecules. A control bead may be functionalized with about10 to about 25 different nucleic acid molecules. A control bead may befunctionalized with about 20 to about 50 different nucleic acidmolecules. A control bead may be functionalized with about 25 to about75 different nucleic acid molecules. A control bead may befunctionalized with about 50 to about 100 different nucleic acidmolecules. A control bead may be functionalized with about 75 to about150 different nucleic acid molecules. A control bead may befunctionalized with about 100 to about 500 different nucleic acidmolecules. A control bead may be functionalized with about 250 to about1000 different nucleic acid molecules. A control bead may befunctionalized with about 96 different nucleic acid molecules.

A control bead may comprise (e.g., may be functionalized with) two ormore different nucleic acid sequences in two or more specific ratios(see e.g., FIG. 10, illustrating that one or more synthetic cells caneach be functionalized with one or more different nucleic acidmolecules). For example, a control bead may be functionalized with twodifferent nucleic acid molecules (e.g., nucleic acid moleculescomprising different nucleic acid sequences or are otherwise differentlymodified, such as those comprising different modifications at their 3′and/or 5′ termini), wherein the bead may be functionalized with the twodifferent nucleic acid molecules in a specific ratio. Such rations canvary widely, depending on application of the control bead or syntheticcell. As another example, a first control bead may be functionalizedwith 96 different nucleic acid sequences (e.g., genes), and a secondcontrol bead may be functionalized with another 96 different nucleicacid sequences (e.g., genes), wherein the 96 different nucleic acidsequences of the first control bead are different (e.g., comprisedifferent nucleic acid sequences) compared to the 96 different nucleicacid sequences (e.g., genes) of the second control bead. Such first andsecond control beads may be used as quality control elements in cellanalysis kits.

A control bead may comprise a first nucleic acid molecule and a secondnucleic acid molecule. The first nucleic acid molecule and the secondnucleic acid molecule may be attached to the control bead in a specificratio. The ratio of the first nucleic acid molecule to the secondnucleic acid molecule may be between about 10⁻³ to about 10³. The ratioof the first nucleic acid molecule to the second nucleic acid moleculemay be between about 10⁻² to about 10². The ratio of the first nucleicacid molecule to the second nucleic acid molecule may be between about10⁻¹ to about 10. The ratio of the first nucleic acid molecule to thesecond nucleic acid molecule may be between about 1 to about 5. Theratio of the first nucleic acid molecule to the second nucleic acidmolecule may be at least about 10⁻¹. The ratio of the first nucleic acidmolecule to the second nucleic acid molecule may be at least about 10⁻¹.The ratio of the first nucleic acid molecule to the second nucleic acidmolecule may be at least about 0.5. The ratio of the first nucleic acidmolecule to the second nucleic acid molecule may be at least about 1.The ratio of the first nucleic acid molecule to the second nucleic acidmolecule may be at least about 5. The ratio of the first nucleic acidmolecule to the second nucleic acid molecule may be at least about 10.The ratio of the first nucleic acid molecule to the second nucleic acidmolecule may be at least about 10².

The compositions, methods and kits of the present disclosure maycomprise a first control bead and a second control bead. The firstcontrol bead and the second control bead may be used and/or be presentin a specific ratio. The ratio of the first control bead to the secondcontrol bead may be between about 10⁻³ to about 10³. The ratio of thefirst control bead to the second control bead may be between about 10⁻²to about 10². The ratio of the first control bead to the second controlbead may be between about 10⁻¹ to about 10³. The ratio of the firstcontrol bead to the second control bead may be between about 1 to about5. The ratio of the first control bead to the second control bead may beat least about 10⁻¹. The ratio of the first control bead to the secondcontrol bead may be at least about 10⁻¹. The ratio of the first controlbead to the second control bead may be at least about 0.5. The ratio ofthe first control bead to the second control bead may be at leastabout 1. The ratio of the first control bead to the second control beadmay be at least about 5. The ratio of the first control bead to thesecond control bead may be at least about 10. The ratio of the firstcontrol bead to the second control bead may be at least about 10².

The herein described compositions, methods and kits may comprise one ormore sets of control beads. A set of control beads may comprise one ormore subset of control beads, e.g., a first subset of control beads anda second subset of control beads. A first set (or subset) of controlbeads and a second set (or subset) of control beads may be present insuch a composition, methods, and/or kit in one or more ratios. Such aratio may be at least about 1:0.001, 1:0.01, 1:0.1, 1:0.2, 1:0.3, 1:0.4,1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4,1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100,1:1000, or more. Such a ratio may be at most about 1:1000, 1;100, 1:90,1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5,1:4, 1:3, 1:2, 1:1.9, 1:1.8, 1:1.7, 1:1.6, 1:1.5, 1:1.4, 1:1.3, 1:1.2,1:1.1, 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2,1:0.1, 1:0.01, 1:0.001, or less.

The compositions, methods and kits of the present disclosure maycomprise control beads. As described herein, such control beads maycomprise two or more different control beads, wherein the two or moredifferent control beads may be used or are present in various ratios asfurther described herein. The two or more different control beads mayeach comprise (or be functionalized with) two or more different nucleicacid molecules, wherein such two or more different nucleic acidmolecules of a synthetic cell may be used or are present in variousratios within the synthetic cell or on the surface of the control bead.Thus, the compositions, methods and kits of the present disclosure mayuse such control beads in a variety of ratios and amounts. For example,a composition, method, and/or kit may comprise two control beads (or twosets or subsets of control beads), wherein each of the two control beadscomprises or is functionalized with about 96 different nucleic acidmolecules (e.g., RNA molecules). Said two control beads may be used incertain, defined ratios. In some cases, such ratio is about 1:2.Similarly, each of the about 96 different nucleic acid molecules thatthe control bead may be functionalized with may be present within(and/or on the surface of) said control bead or on the surface of saidcontrol bead in certain, defined ratios. Said two control beads may beused in a method or kit as described herein.

A control bead disclosed herein may mimic single cell behavior in singlecell processing experiments. As described herein, said control bead maybe a bead comprising one or more known sequences. The bead may be a gelbead. The control bead may be introduced into a cell sample and haveapproximately the same or substantially the same size as other cells inthe cell sample. The control bead may be carried through the entireworkflow of a single cell processing experiment with the cell sample.After sequencing, the one or more known sequences in the control beadmay be identified and analyzed to determine various characteristics ofthe single cell processing experiment, such as the effectiveness or theefficiency of the library preparation process or the sequencing process.Thus, the control beads (e.g., synthetic cells) described herein may beused in library preparation processes and/or in sequencing experimentsand may be used as control standards that allow such processes to beperformed with increased accuracy and efficiency compared toconventional methodologies. For example, control beads may beparticularly useful in the analysis and processing of biological samplesthat contain cells of more than one species, such as human and mousecells (e.g., barnyard experiments). For example, a control bead of thepresent disclosure may be used to account for PCR chimeras that may beproduced during such barnyard experiments.

The compositions, methods and kits of the present disclosure maycomprise barcode-containing beads and/or control beads, or anycombination thereof (e.g., synthetic cells) in partitions (e.g.,droplets or wells) for analyzing biological samples (e.g., single-cellanalysis). The control beads may act as a quality control element and/orinternal standard to improve accuracy and efficiency of single-cellanalyses, and address the shortcomings of current methodologies asfurther described herein. For example, a control bead of the presentdisclosure may be used to determine a doublet rate.

Reagents

In accordance with certain aspects, analyte carriers may be partitionedalong with lysis reagents in order to release the contents of theanalyte carriers within the partition. In such cases, the lysis agentscan be contacted with the analyte carrier suspension concurrently with,or immediately prior to, the introduction of the analyte carriers intothe partitioning junction/droplet generation zone (e.g., junction 210),such as through an additional channel or channels upstream of thechannel junction. In accordance with other aspects, additionally oralternatively, analyte carriers may be partitioned along with otherreagents, as will be described further below.

FIG. 3 shows an example of a microfluidic channel structure 300 forco-partitioning analyte carriers and reagents. The channel structure 300can include channel segments 301, 302, 304, 306 and 308. Channelsegments 301 and 302 communicate at a first channel junction 309.Channel segments 302, 304, 306, and 308 communicate at a second channeljunction 310.

In an example operation, the channel segment 301 may transport anaqueous fluid 312 that includes a plurality of analyte carriers 314along the channel segment 301 into the second junction 310. As analternative or in addition to, channel segment 301 may transport beads(e.g., gel beads). The beads may comprise barcode molecules.

For example, the channel segment 301 may be connected to a reservoircomprising an aqueous suspension of analyte carriers 314. Upstream of,and immediately prior to reaching, the second junction 310, the channelsegment 301 may meet the channel segment 302 at the first junction 309.The channel segment 302 may transport a plurality of reagents 315 (e.g.,lysis agents) suspended in the aqueous fluid 312 along the channelsegment 302 into the first junction 309. For example, the channelsegment 302 may be connected to a reservoir comprising the reagents 315.After the first junction 309, the aqueous fluid 312 in the channelsegment 301 can carry both the analyte carriers 314 and the reagents 315towards the second junction 310. In some instances, the aqueous fluid312 in the channel segment 301 can include one or more reagents, whichcan be the same or different reagents as the reagents 315. A secondfluid 316 that is immiscible with the aqueous fluid 312 (e.g., oil) canbe delivered to the second junction 310 from each of channel segments304 and 306. Upon meeting of the aqueous fluid 312 from the channelsegment 301 and the second fluid 316 from each of channel segments 304and 306 at the second channel junction 310, the aqueous fluid 312 can bepartitioned as discrete droplets 318 in the second fluid 316 and flowaway from the second junction 310 along channel segment 308. The channelsegment 308 may deliver the discrete droplets 318 to an outlet reservoirfluidly coupled to the channel segment 308, where they may be harvested.

The second fluid 316 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resulting droplets318.

A discrete droplet generated may include an individual analyte carrier314 and/or one or more reagents 315. In some instances, a discretedroplet generated may include a barcode carrying bead (not shown), suchas via other microfluidics structures described elsewhere herein. Insome instances, a discrete droplet may be unoccupied (e.g., no reagents,no analyte carriers).

Beneficially, when lysis reagents and analyte carriers areco-partitioned, the lysis reagents can facilitate the release of thecontents of the analyte carriers within the partition. The contentsreleased in a partition may remain discrete from the contents of otherpartitions.

As will be appreciated, the channel segments described herein may becoupled to any of a variety of different fluid sources or receivingcomponents, including reservoirs, tubing, manifolds, or fluidiccomponents of other systems. As will be appreciated, the microfluidicchannel structure 300 may have other geometries. For example, amicrofluidic channel structure can have more than two channel junctions.For example, a microfluidic channel structure can have 2, 3, 4, 5channel segments or more each carrying the same or different types ofbeads, reagents, and/or analyte carriers that meet at a channeljunction. Fluid flow in each channel segment may be controlled tocontrol the partitioning of the different elements into droplets. Fluidmay be directed flow along one or more channels or reservoirs via one ormore fluid flow units. A fluid flow unit can comprise compressors (e.g.,providing positive pressure), pumps (e.g., providing negative pressure),actuators, and the like to control flow of the fluid. Fluid may also orotherwise be controlled via applied pressure differentials, centrifugalforce, electrokinetic pumping, vacuum, capillary or gravity flow, or thelike.

Examples of lysis agents include bioactive reagents, such as lysisenzymes that are used for lysis of different cell types, e.g., grampositive or negative bacteria, plants, yeast, mammalian, etc., such aslysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase,and a variety of other lysis enzymes available from, e.g.,Sigma-Aldrich, Inc. (St Louis, Mo.), as well as other commerciallyavailable lysis enzymes. Other lysis agents may additionally oralternatively be co-partitioned with the analyte carriers to cause therelease of the analyte carriers's contents into the partitions. Forexample, in some cases, surfactant-based lysis solutions may be used tolyse cells, although these may be less desirable for emulsion basedsystems where the surfactants can interfere with stable emulsions. Insome cases, lysis solutions may include non-ionic surfactants such as,for example, TritonX-100 and Tween 20. In some cases, lysis solutionsmay include ionic surfactants such as, for example, sarcosyl and sodiumdodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanicalcellular disruption may also be used in certain cases, e.g.,non-emulsion based partitioning such as encapsulation of analytecarriers that may be in addition to or in place of droplet partitioning,where any pore size of the encapsulate is sufficiently small to retainnucleic acid fragments of a given size, following cellular disruption.

Alternatively or in addition to the lysis agents co-partitioned with theanalyte carriers described above, other reagents can also beco-partitioned with the analyte carriers, including, for example, DNaseand RNase inactivating agents or inhibitors, such as proteinase K,chelating agents, such as EDTA, and other reagents employed in removingor otherwise reducing negative activity or impact of different celllysate components on subsequent processing of nucleic acids. Inaddition, in the case of encapsulated analyte carriers, the analytecarriers may be exposed to an appropriate stimulus to release theanalyte carriers or their contents from a co-partitioned microcapsule.For example, in some cases, a chemical stimulus may be co-partitionedalong with an encapsulated analyte carrier to allow for the degradationof the microcapsule and release of the cell or its contents into thelarger partition. In some cases, this stimulus may be the same as thestimulus described elsewhere herein for release of nucleic acidmolecules (e.g., oligonucleotides) from their respective microcapsule(e.g., bead). In alternative aspects, this may be a different andnon-overlapping stimulus, in order to allow an encapsulated analytecarrier to be released into a partition at a different time from therelease of nucleic acid molecules into the same partition.

Additional reagents may also be co-partitioned with the analytecarriers, such as endonucleases to fragment an analyte carrier's DNA,DNA polymerase enzymes and dNTPs used to amplify the analyte carrier'snucleic acid fragments and to attach the barcode molecular tags to theamplified fragments. Other enzymes may be co-partitioned, includingwithout limitation, polymerase, transposase, ligase, proteinase K,DNAse, etc. Additional reagents may also include reverse transcriptaseenzymes, including enzymes with terminal transferase activity, primersand oligonucleotides, and switch oligonucleotides (also referred toherein as “switch oligos” or “template switching oligonucleotides”)which can be used for template switching. In some cases, templateswitching can be used to increase the length of a cDNA. In some cases,template switching can be used to append a predefined nucleic acidsequence to the cDNA. In an example of template switching, cDNA can begenerated from reverse transcription of a template, e.g., cellular mRNA,where a reverse transcriptase with terminal transferase activity can addadditional nucleotides, e.g., polyC, to the cDNA in a templateindependent manner. Switch oligos can include sequences complementary tothe additional nucleotides, e.g., polyG. The additional nucleotides(e.g., polyC) on the cDNA can hybridize to the additional nucleotides(e.g., polyG) on the switch oligo, whereby the switch oligo can be usedby the reverse transcriptase as template to further extend the cDNA.Template switching oligonucleotides may comprise a hybridization regionand a template region. The hybridization region can comprise anysequence capable of hybridizing to the target. In some cases, aspreviously described, the hybridization region comprises a series of Gbases to complement the overhanging C bases at the 3′ end of a cDNAmolecule. The series of G bases may comprise 1 G base, 2 G bases, 3 Gbases, 4 G bases, 5 G bases or more than 5 G bases. The templatesequence can comprise any sequence to be incorporated into the cDNA. Insome cases, the template region comprises at least 1 (e.g., at least 2,3, 4, 5 or more) tag sequences and/or functional sequences. Switcholigos may comprise deoxyribonucleic acids; ribonucleic acids; modifiednucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA),inverted dT, 5-Methyl dC, 2′-deoxyInosine, Super T(5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C,Fluoro U, Fluoro A, and Fluoro G), or any combination.

In some cases, the length of a switch oligo may be at least about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides or longer.

In some cases, the length of a switch oligo may be at most about 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or250 nucleotides.

Once the contents of the cells are released into their respectivepartitions, the macromolecular components (e.g., macromolecularconstituents of analyte carriers, such as RNA, DNA, or proteins)contained therein may be further processed within the partitions. Inaccordance with the methods and systems described herein, themacromolecular component contents of individual analyte carriers can beprovided with unique identifiers such that, upon characterization ofthose macromolecular components they may be attributed as having beenderived from the same analyte carrier or particles. The ability toattribute characteristics to individual analyte carriers or groups ofanalyte carriers is provided by the assignment of unique identifiersspecifically to an individual analyte carrier or groups of analytecarriers. Unique identifiers, e.g., in the form of nucleic acid barcodescan be assigned or associated with individual analyte carriers orpopulations of analyte carriers, in order to tag or label the analytecarrier's macromolecular components (and as a result, itscharacteristics) with the unique identifiers. These unique identifierscan then be used to attribute the analyte carrier's components andcharacteristics to an individual analyte carrier or group of analytecarriers.

In some aspects, this is performed by co-partitioning the individualanalyte carrier or groups of analyte carriers with the uniqueidentifiers, such as described above (with reference to FIG. 2). In someaspects, the unique identifiers are provided in the form of nucleic acidmolecules (e.g., oligonucleotides) that comprise nucleic acid barcodesequences that may be attached to or otherwise associated with thenucleic acid contents of individual analyte carrier, or to othercomponents of the analyte carrier, and particularly to fragments ofthose nucleic acids. The nucleic acid molecules are partitioned suchthat as between nucleic acid molecules in a given partition, the nucleicacid barcode sequences contained therein are the same, but as betweendifferent partitions, the nucleic acid molecule can, and do havediffering barcode sequences, or at least represent a large number ofdifferent barcode sequences across all of the partitions in a givenanalysis. In some aspects, only one nucleic acid barcode sequence can beassociated with a given partition, although in some cases, two or moredifferent barcode sequences may be present.

The nucleic acid barcode sequences can include from about 6 to about 20or more nucleotides within the sequence of the nucleic acid molecules(e.g., oligonucleotides). The nucleic acid barcode sequences can includefrom about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or morenucleotides. In some cases, the length of a barcode sequence may beabout 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotidesor longer. In some cases, the length of a barcode sequence may be atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20nucleotides or longer. In some cases, the length of a barcode sequencemay be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 nucleotides or shorter. These nucleotides may be completelycontiguous, i.e., in a single stretch of adjacent nucleotides, or theymay be separated into two or more separate subsequences that areseparated by 1 or more nucleotides. In some cases, separated barcodesubsequences can be from about 4 to about 16 nucleotides in length. Insome cases, the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16 nucleotides or longer. In some cases, the barcode subsequence maybe at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

The co-partitioned nucleic acid molecules can also comprise otherfunctional sequences useful in the processing of the nucleic acids fromthe co-partitioned analyte carriers. These sequences include, e.g.,targeted or random/universal amplification primer sequences foramplifying the genomic DNA from the individual analyte carriers withinthe partitions while attaching the associated barcode sequences,sequencing primers or primer recognition sites, hybridization or probingsequences, e.g., for identification of presence of the sequences or forpulling down barcoded nucleic acids, or any of a number of otherpotential functional sequences. Other mechanisms of co-partitioningoligonucleotides may also be employed, including, e.g., coalescence oftwo or more droplets, where one droplet contains oligonucleotides, ormicrodispensing of oligonucleotides into partitions, e.g., dropletswithin microfluidic systems.

In an example, microcapsules, such as beads, are provided that eachinclude large numbers of the above described barcoded nucleic acidmolecules (e.g., barcoded oligonucleotides) releasably attached to thebeads, where all of the nucleic acid molecules attached to a particularbead will include the same nucleic acid barcode sequence, but where alarge number of diverse barcode sequences are represented across thepopulation of beads used. In some embodiments, hydrogel beads, e.g.,comprising polyacrylamide polymer matrices, are used as a solid supportand delivery vehicle for the nucleic acid molecules into the partitions,as they are capable of carrying large numbers of nucleic acid molecules,and may be configured to release those nucleic acid molecules uponexposure to a particular stimulus, as described elsewhere herein. Insome cases, the population of beads provides a diverse barcode sequencelibrary that includes at least about 1,000 different barcode sequences,at least about 5,000 different barcode sequences, at least about 10,000different barcode sequences, at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences, or more. Additionally, each bead can be provided withlarge numbers of nucleic acid (e.g., oligonucleotide) moleculesattached. In particular, the number of molecules of nucleic acidmolecules including the barcode sequence on an individual bead can be atleast about 1,000 nucleic acid molecules, at least about 5,000 nucleicacid molecules, at least about 10,000 nucleic acid molecules, at leastabout 50,000 nucleic acid molecules, at least about 100,000 nucleic acidmolecules, at least about 500,000 nucleic acids, at least about1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acidmolecules, at least about 10,000,000 nucleic acid molecules, at leastabout 50,000,000 nucleic acid molecules, at least about 100,000,000nucleic acid molecules, at least about 250,000,000 nucleic acidmolecules and in some cases at least about 1 billion nucleic acidmolecules, or more. Nucleic acid molecules of a given bead can includeidentical (or common) barcode sequences, different barcode sequences, ora combination of both. Nucleic acid molecules of a given bead caninclude multiple sets of nucleic acid molecules. Nucleic acid moleculesof a given set can include identical barcode sequences. The identicalbarcode sequences can be different from barcode sequences of nucleicacid molecules of another set.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least about 1,000 different barcode sequences, at leastabout 5,000 different barcode sequences, at least about 10,000 differentbarcode sequences, at least at least about 50,000 different barcodesequences, at least about 100,000 different barcode sequences, at leastabout 1,000,000 different barcode sequences, at least about 5,000,000different barcode sequences, or at least about 10,000,000 differentbarcode sequences. Additionally, each partition of the population caninclude at least about 1,000 nucleic acid molecules, at least about5,000 nucleic acid molecules, at least about 10,000 nucleic acidmolecules, at least about 50,000 nucleic acid molecules, at least about100,000 nucleic acid molecules, at least about 500,000 nucleic acids, atleast about 1,000,000 nucleic acid molecules, at least about 5,000,000nucleic acid molecules, at least about 10,000,000 nucleic acidmolecules, at least about 50,000,000 nucleic acid molecules, at leastabout 100,000,000 nucleic acid molecules, at least about 250,000,000nucleic acid molecules and in some cases at least about 1 billionnucleic acid molecules.

In some cases, it may be desirable to incorporate multiple differentbarcodes within a given partition, either attached to a single ormultiple beads within the partition. For example, in some cases, amixed, but known set of barcode sequences may provide greater assuranceof identification in the subsequent processing, e.g., by providing astronger address or attribution of the barcodes to a given partition, asa duplicate or independent confirmation of the output from a givenpartition.

The nucleic acid molecules (e.g., oligonucleotides) are releasable fromthe beads upon the application of a particular stimulus to the beads. Insome cases, the stimulus may be a photo-stimulus, e.g., through cleavageof a photo-labile linkage that releases the nucleic acid molecules. Inother cases, a thermal stimulus may be used, where elevation of thetemperature of the beads environment will result in cleavage of alinkage or other release of the nucleic acid molecules form the beads.In still other cases, a chemical stimulus can be used that cleaves alinkage of the nucleic acid molecules to the beads, or otherwise resultsin release of the nucleic acid molecules from the beads. In one case,such compositions include the polyacrylamide matrices described abovefor encapsulation of analyte carriers, and may be degraded for releaseof the attached nucleic acid molecules through exposure to a reducingagent, such as DTT.

In some aspects, provided are systems and methods for controlledpartitioning. Droplet size may be controlled by adjusting certaingeometric features in channel architecture (e.g., microfluidics channelarchitecture). For example, an expansion angle, width, and/or length ofa channel may be adjusted to control droplet size.

FIG. 4 shows an example of a microfluidic channel structure for thecontrolled partitioning of beads into discrete droplets. A channelstructure 400 can include a channel segment 402 communicating at achannel junction 406 (or intersection) with a reservoir 404. Thereservoir 404 can be a chamber. Any reference to “reservoir,” as usedherein, can also refer to a “chamber.” In operation, an aqueous fluid408 that includes suspended beads 412 may be transported along thechannel segment 402 into the junction 406 to meet a second fluid 410that is immiscible with the aqueous fluid 408 in the reservoir 404 tocreate droplets 416, 418 of the aqueous fluid 408 flowing into thereservoir 404. At the junction 406 where the aqueous fluid 408 and thesecond fluid 410 meet, droplets can form based on factors such as thehydrodynamic forces at the junction 406, flow rates of the two fluids408, 410, fluid properties, and certain geometric parameters (e.g., w,h₀, α, etc.) of the channel structure 400. A plurality of droplets canbe collected in the reservoir 404 by continuously injecting the aqueousfluid 408 from the channel segment 402 through the junction 406.

A discrete droplet generated may include a bead (e.g., as in occupieddroplets 416). Alternatively, a discrete droplet generated may includemore than one bead. Alternatively, a discrete droplet generated may notinclude any beads (e.g., as in unoccupied droplet 418). In someinstances, a discrete droplet generated may contain one or more analytecarriers, as described elsewhere herein. In some instances, a discretedroplet generated may comprise one or more reagents, as describedelsewhere herein.

In some instances, the aqueous fluid 408 can have a substantiallyuniform concentration or frequency of beads 412. The beads 412 can beintroduced into the channel segment 402 from a separate channel (notshown in FIG. 4). The frequency of beads 412 in the channel segment 402may be controlled by controlling the frequency in which the beads 412are introduced into the channel segment 402 and/or the relative flowrates of the fluids in the channel segment 402 and the separate channel.In some instances, the beads can be introduced into the channel segment402 from a plurality of different channels, and the frequency controlledaccordingly.

In some instances, the aqueous fluid 408 in the channel segment 402 cancomprise analyte carriers (e.g., described with reference to FIGS. 1 and2). In some instances, the aqueous fluid 408 can have a substantiallyuniform concentration or frequency of analyte carriers. As with thebeads, the analyte carriers can be introduced into the channel segment402 from a separate channel. The frequency or concentration of theanalyte carriers in the aqueous fluid 408 in the channel segment 402 maybe controlled by controlling the frequency in which the analyte carriersare introduced into the channel segment 402 and/or the relative flowrates of the fluids in the channel segment 402 and the separate channel.In some instances, the analyte carriers can be introduced into thechannel segment 402 from a plurality of different channels, and thefrequency controlled accordingly. In some instances, a first separatechannel can introduce beads and a second separate channel can introduceanalyte carriers into the channel segment 402. The first separatechannel introducing the beads may be upstream or downstream of thesecond separate channel introducing the analyte carriers.

The second fluid 410 can comprise an oil, such as a fluorinated oil,that includes a fluorosurfactant for stabilizing the resulting droplets,for example, inhibiting subsequent coalescence of the resultingdroplets.

In some instances, the second fluid 410 may not be subjected to and/ordirected to any flow in or out of the reservoir 404. For example, thesecond fluid 410 may be substantially stationary in the reservoir 404.In some instances, the second fluid 410 may be subjected to flow withinthe reservoir 404, but not in or out of the reservoir 404, such as viaapplication of pressure to the reservoir 404 and/or as affected by theincoming flow of the aqueous fluid 408 at the junction 406.Alternatively, the second fluid 410 may be subjected and/or directed toflow in or out of the reservoir 404. For example, the reservoir 404 canbe a channel directing the second fluid 410 from upstream to downstream,transporting the generated droplets.

The channel structure 400 at or near the junction 406 may have certaingeometric features that at least partly determine the sizes of thedroplets formed by the channel structure 400. The channel segment 402can have a height, h₀ and width, w, at or near the junction 406. By wayof example, the channel segment 402 can comprise a rectangularcross-section that leads to a reservoir 404 having a wider cross-section(such as in width or diameter). Alternatively, the cross-section of thechannel segment 402 can be other shapes, such as a circular shape,trapezoidal shape, polygonal shape, or any other shapes. The top andbottom walls of the reservoir 404 at or near the junction 406 can beinclined at an expansion angle, α. The expansion angle, α, allows thetongue (portion of the aqueous fluid 408 leaving channel segment 402 atjunction 406 and entering the reservoir 404 before droplet formation) toincrease in depth and facilitate decrease in curvature of theintermediately formed droplet. Droplet size may decrease with increasingexpansion angle. The resulting droplet radius, R_(d), may be predictedby the following equation for the aforementioned geometric parameters ofh₀, w, and α:

$R_{d} \approx {0.44\left( {1 + {2.2\sqrt{\tan\mspace{14mu}\alpha}\frac{w}{h_{0}}}} \right)\frac{h_{0}}{\sqrt{\tan\mspace{14mu}\alpha}}}$

By way of example, for a channel structure with w=21 μm, h=21 μm, andα=3°, the predicted droplet size is 121 μm. In another example, for achannel structure with w=25 h=25 μm, and α=5°, the predicted dropletsize is 123 μm. In another example, for a channel structure with w=28μm, h=28 μm, and α=7°, the predicted droplet size is 124 μm.

In some instances, the expansion angle, α, may be between a range offrom about 0.5° to about 4°, from about 0.1° to about 10°, or from about0° to about 90°. For example, the expansion angle can be at least about0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°,4°, 5°, 6° 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°,60°, 65°, 70°, 75°, 80°, 85°, or higher. In some instances, theexpansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°,82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°,20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.In some instances, the width, w, can be between a range of from about100 micrometers (μall) to about 500 μm. In some instances, the width, w,can be between a range of from about 10 μm to about 200 μm.Alternatively, the width can be less than about 10 μm. Alternatively,the width can be greater than about 500 μm. In some instances, the flowrate of the aqueous fluid 408 entering the junction 406 can be betweenabout 0.04 microliters (μL)/minute (min) and about 40 μL/min. In someinstances, the flow rate of the aqueous fluid 408 entering the junction406 can be between about 0.01 microliters (μL)/minute (min) and about100 μL/min. Alternatively, the flow rate of the aqueous fluid 408entering the junction 406 can be less than about 0.01 μL/min.Alternatively, the flow rate of the aqueous fluid 408 entering thejunction 406 can be greater than about 40 μL/min, such as 45 μL/min, 50μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75 μL/min, 80μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110 μL/min, 120μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. At lower flowrates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 408 entering the junction 406.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

The throughput of droplet generation can be increased by increasing thepoints of generation, such as increasing the number of junctions (e.g.,junction 406) between aqueous fluid 408 channel segments (e.g., channelsegment 402) and the reservoir 404. Alternatively or in addition, thethroughput of droplet generation can be increased by increasing the flowrate of the aqueous fluid 408 in the channel segment 402.

FIG. 5 shows an example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 500 can comprise a plurality of channel segments 502 and areservoir 504. Each of the plurality of channel segments 502 may be influid communication with the reservoir 504. The channel structure 500can comprise a plurality of channel junctions 506 between the pluralityof channel segments 502 and the reservoir 504. Each channel junction canbe a point of droplet generation. The channel segment 402 from thechannel structure 400 in FIG. 4 and any description to the componentsthereof may correspond to a given channel segment of the plurality ofchannel segments 502 in channel structure 500 and any description to thecorresponding components thereof. The reservoir 404 from the channelstructure 400 and any description to the components thereof maycorrespond to the reservoir 504 from the channel structure 500 and anydescription to the corresponding components thereof.

Each channel segment of the plurality of channel segments 502 maycomprise an aqueous fluid 508 that includes suspended beads 512. Thereservoir 504 may comprise a second fluid 510 that is immiscible withthe aqueous fluid 508. In some instances, the second fluid 510 may notbe subjected to and/or directed to any flow in or out of the reservoir504. For example, the second fluid 510 may be substantially stationaryin the reservoir 504. In some instances, the second fluid 510 may besubjected to flow within the reservoir 504, but not in or out of thereservoir 504, such as via application of pressure to the reservoir 504and/or as affected by the incoming flow of the aqueous fluid 508 at thejunctions. Alternatively, the second fluid 510 may be subjected and/ordirected to flow in or out of the reservoir 504. For example, thereservoir 504 can be a channel directing the second fluid 510 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 508 that includes suspended beads 512may be transported along the plurality of channel segments 502 into theplurality of junctions 506 to meet the second fluid 510 in the reservoir504 to create droplets 516, 518. A droplet may form from each channelsegment at each corresponding junction with the reservoir 504. At thejunction where the aqueous fluid 508 and the second fluid 510 meet,droplets can form based on factors such as the hydrodynamic forces atthe junction, flow rates of the two fluids 508, 510, fluid properties,and certain geometric parameters (e.g., w, h₀, α, etc.) of the channelstructure 500, as described elsewhere herein. A plurality of dropletscan be collected in the reservoir 504 by continuously injecting theaqueous fluid 508 from the plurality of channel segments 502 through theplurality of junctions 506. Throughput may significantly increase withthe parallel channel configuration of channel structure 500. Forexample, a channel structure having five inlet channel segmentscomprising the aqueous fluid 508 may generate droplets five times asfrequently than a channel structure having one inlet channel segment,provided that the fluid flow rate in the channel segments aresubstantially the same. The fluid flow rate in the different inletchannel segments may or may not be substantially the same. A channelstructure may have as many parallel channel segments as is practical andallowed for the size of the reservoir. For example, the channelstructure may have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 500, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantiallyparallel channel segments.

The geometric parameters, w, h₀, and α, may or may not be uniform foreach of the channel segments in the plurality of channel segments 502.For example, each channel segment may have the same or different widthsat or near its respective channel junction with the reservoir 504. Forexample, each channel segment may have the same or different height ator near its respective channel junction with the reservoir 504. Inanother example, the reservoir 504 may have the same or differentexpansion angle at the different channel junctions with the plurality ofchannel segments 502. When the geometric parameters are uniform,beneficially, droplet size may also be controlled to be uniform evenwith the increased throughput. In some instances, when it is desirableto have a different distribution of droplet sizes, the geometricparameters for the plurality of channel segments 502 may be variedaccordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

FIG. 6 shows another example of a microfluidic channel structure forincreased droplet generation throughput. A microfluidic channelstructure 600 can comprise a plurality of channel segments 602 arrangedgenerally circularly around the perimeter of a reservoir 604. Each ofthe plurality of channel segments 602 may be in fluid communication withthe reservoir 604. The channel structure 600 can comprise a plurality ofchannel junctions 606 between the plurality of channel segments 602 andthe reservoir 604. Each channel junction can be a point of dropletgeneration. The channel segment 402 from the channel structure 400 inFIG. 2 and any description to the components thereof may correspond to agiven channel segment of the plurality of channel segments 602 inchannel structure 600 and any description to the correspondingcomponents thereof. The reservoir 404 from the channel structure 400 andany description to the components thereof may correspond to thereservoir 604 from the channel structure 600 and any description to thecorresponding components thereof.

Each channel segment of the plurality of channel segments 602 maycomprise an aqueous fluid 608 that includes suspended beads 612. Thereservoir 604 may comprise a second fluid 610 that is immiscible withthe aqueous fluid 608. In some instances, the second fluid 610 may notbe subjected to and/or directed to any flow in or out of the reservoir604. For example, the second fluid 610 may be substantially stationaryin the reservoir 604. In some instances, the second fluid 610 may besubjected to flow within the reservoir 604, but not in or out of thereservoir 604, such as via application of pressure to the reservoir 604and/or as affected by the incoming flow of the aqueous fluid 608 at thejunctions. Alternatively, the second fluid 610 may be subjected and/ordirected to flow in or out of the reservoir 604. For example, thereservoir 604 can be a channel directing the second fluid 610 fromupstream to downstream, transporting the generated droplets.

In operation, the aqueous fluid 608 that includes suspended beads 612may be transported along the plurality of channel segments 602 into theplurality of junctions 606 to meet the second fluid 610 in the reservoir604 to create a plurality of droplets 616. A droplet may form from eachchannel segment at each corresponding junction with the reservoir 604.At the junction where the aqueous fluid 608 and the second fluid 610meet, droplets can form based on factors such as the hydrodynamic forcesat the junction, flow rates of the two fluids 608, 610, fluidproperties, and certain geometric parameters (e.g., widths and heightsof the channel segments 602, expansion angle of the reservoir 604, etc.)of the channel structure 600, as described elsewhere herein. A pluralityof droplets can be collected in the reservoir 604 by continuouslyinjecting the aqueous fluid 608 from the plurality of channel segments602 through the plurality of junctions 606. Throughput may significantlyincrease with the substantially parallel channel configuration of thechannel structure 600. A channel structure may have as manysubstantially parallel channel segments as is practical and allowed forby the size of the reservoir. For example, the channel structure mayhave at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 1500, 5000 or more parallel or substantially parallel channelsegments. The plurality of channel segments may be substantially evenlyspaced apart, for example, around an edge or perimeter of the reservoir.Alternatively, the spacing of the plurality of channel segments may beuneven.

The reservoir 604 may have an expansion angle, α (not shown in FIG. 6)at or near each channel junction. Each channel segment of the pluralityof channel segments 602 may have a width, w, and a height, h₀, at ornear the channel junction. The geometric parameters, w, h₀, and α, mayor may not be uniform for each of the channel segments in the pluralityof channel segments 602. For example, each channel segment may have thesame or different widths at or near its respective channel junction withthe reservoir 604. For example, each channel segment may have the sameor different height at or near its respective channel junction with thereservoir 604.

The reservoir 604 may have the same or different expansion angle at thedifferent channel junctions with the plurality of channel segments 602.For example, a circular reservoir (as shown in FIG. 6) may have aconical, dome-like, or hemispherical ceiling (e.g., top wall) to providethe same or substantially same expansion angle for each channel segments602 at or near the plurality of channel junctions 606. When thegeometric parameters are uniform, beneficially, resulting droplet sizemay be controlled to be uniform even with the increased throughput. Insome instances, when it is desirable to have a different distribution ofdroplet sizes, the geometric parameters for the plurality of channelsegments 602 may be varied accordingly.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size. The beads and/or analytecarrier injected into the droplets may or may not have uniform size.

FIG. 7A shows a cross-section view of another example of a microfluidicchannel structure with a geometric feature for controlled partitioning.A channel structure 700 can include a channel segment 702 communicatingat a channel junction 706 (or intersection) with a reservoir 704. Insome instances, the channel structure 700 and one or more of itscomponents can correspond to the channel structure 100 and one or moreof its components. FIG. 7B shows a perspective view of the channelstructure 700 of FIG. 7A.

An aqueous fluid 712 comprising a plurality of particles 716 may betransported along the channel segment 702 into the junction 706 to meeta second fluid 714 (e.g., oil, etc.) that is immiscible with the aqueousfluid 712 in the reservoir 704 to create droplets 720 of the aqueousfluid 712 flowing into the reservoir 704. At the junction 706 where theaqueous fluid 712 and the second fluid 714 meet, droplets can form basedon factors such as the hydrodynamic forces at the junction 706, relativeflow rates of the two fluids 712, 714, fluid properties, and certaingeometric parameters (e.g., Δh, etc.) of the channel structure 700. Aplurality of droplets can be collected in the reservoir 704 bycontinuously injecting the aqueous fluid 712 from the channel segment702 at the junction 706.

A discrete droplet generated may comprise one or more particles of theplurality of particles 716. As described elsewhere herein, a particlemay be any particle, such as a bead, cell bead, gel bead, analytecarrier, macromolecular constituents of analyte carrier, or otherparticles. Alternatively, a discrete droplet generated may not includeany particles.

In some instances, the aqueous fluid 712 can have a substantiallyuniform concentration or frequency of particles 716. As describedelsewhere herein (e.g., with reference to FIG. 4), the particles 716(e.g., beads) can be introduced into the channel segment 702 from aseparate channel (not shown in FIG. 7). The frequency of particles 716in the channel segment 702 may be controlled by controlling thefrequency in which the particles 716 are introduced into the channelsegment 702 and/or the relative flow rates of the fluids in the channelsegment 702 and the separate channel. In some instances, the particles716 can be introduced into the channel segment 702 from a plurality ofdifferent channels, and the frequency controlled accordingly. In someinstances, different particles may be introduced via separate channels.For example, a first separate channel can introduce beads and a secondseparate channel can introduce analyte carriers into the channel segment702. The first separate channel introducing the beads may be upstream ordownstream of the second separate channel introducing the analytecarriers.

In some instances, the second fluid 714 may not be subjected to and/ordirected to any flow in or out of the reservoir 704. For example, thesecond fluid 714 may be substantially stationary in the reservoir 704.In some instances, the second fluid 714 may be subjected to flow withinthe reservoir 704, but not in or out of the reservoir 704, such as viaapplication of pressure to the reservoir 704 and/or as affected by theincoming flow of the aqueous fluid 712 at the junction 706.Alternatively, the second fluid 714 may be subjected and/or directed toflow in or out of the reservoir 704. For example, the reservoir 704 canbe a channel directing the second fluid 714 from upstream to downstream,transporting the generated droplets.

The channel structure 700 at or near the junction 706 may have certaingeometric features that at least partly determine the sizes and/orshapes of the droplets formed by the channel structure 700. The channelsegment 702 can have a first cross-section height, h₁, and the reservoir704 can have a second cross-section height, h₂. The first cross-sectionheight, h₁, and the second cross-section height, h₂, may be different,such that at the junction 706, there is a height difference of Δh. Thesecond cross-section height, h₂, may be greater than the firstcross-section height, h₁. In some instances, the reservoir maythereafter gradually increase in cross-section height, for example, themore distant it is from the junction 706. In some instances, thecross-section height of the reservoir may increase in accordance withexpansion angle, β, at or near the junction 706. The height difference,Δh, and/or expansion angle, β, can allow the tongue (portion of theaqueous fluid 712 leaving channel segment 702 at junction 706 andentering the reservoir 704 before droplet formation) to increase indepth and facilitate decrease in curvature of the intermediately formeddroplet. For example, droplet size may decrease with increasing heightdifference and/or increasing expansion angle.

The height difference, Δh, can be at least about 1 μm. Alternatively,the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 200, 300, 400, 500 μm or more. Alternatively, theheight difference can be at most about 500, 400, 300, 200, 100, 90, 80,70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μm or less. In some instances, theexpansion angle, β, may be between a range of from about 0.5° to about4°, from about 0.1° to about 10°, or from about 0° to about 90°. Forexample, the expansion angle can be at least about 0.01°, 0.1°, 0.2°,0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°,8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°,75°, 80°, 85°, or higher. In some instances, the expansion angle can beat most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°,70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°,7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

In some instances, the flow rate of the aqueous fluid 712 entering thejunction 706 can be between about 0.04 microliters (μL)/minute (min) andabout 40 μL/min. In some instances, the flow rate of the aqueous fluid712 entering the junction 706 can be between about 0.01 microliters(μL)/minute (min) and about 100 μL/min. Alternatively, the flow rate ofthe aqueous fluid 712 entering the junction 706 can be less than about0.01 μL/min. Alternatively, the flow rate of the aqueous fluid 712entering the junction 706 can be greater than about 40 μL/min, such as45 μL/min, 50 μL/min, 55 μL/min, 60 μL/min, 65 μL/min, 70 μL/min, 75μL/min, 80 μL/min, 85 μL/min, 90 μL/min, 95 μL/min, 100 μL/min, 110μL/min, 120 μL/min, 130 μL/min, 140 μL/min, 150 μL/min, or greater. Atlower flow rates, such as flow rates of about less than or equal to 10microliters/minute, the droplet radius may not be dependent on the flowrate of the aqueous fluid 712 entering the junction 706. The secondfluid 714 may be stationary, or substantially stationary, in thereservoir 704. Alternatively, the second fluid 714 may be flowing, suchas at the above flow rates described for the aqueous fluid 712.

In some instances, at least about 50% of the droplets generated can haveuniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the dropletsgenerated can have uniform size. Alternatively, less than about 50% ofthe droplets generated can have uniform size.

While FIGS. 7A and 7B illustrate the height difference, Δh, being abruptat the junction 706 (e.g., a step increase), the height difference mayincrease gradually (e.g., from about 0 μm to a maximum heightdifference). Alternatively, the height difference may decrease gradually(e.g., taper) from a maximum height difference. A gradual increase ordecrease in height difference, as used herein, may refer to a continuousincremental increase or decrease in height difference, wherein an anglebetween any one differential segment of a height profile and animmediately adjacent differential segment of the height profile isgreater than 90°. For example, at the junction 706, a bottom wall of thechannel and a bottom wall of the reservoir can meet at an angle greaterthan 90°. Alternatively or in addition, a top wall (e.g., ceiling) ofthe channel and a top wall (e.g., ceiling) of the reservoir can meet anangle greater than 90°. A gradual increase or decrease may be linear ornon-linear (e.g., exponential, sinusoidal, etc.). Alternatively or inaddition, the height difference may variably increase and/or decreaselinearly or non-linearly. While FIGS. 7A and 7B illustrate the expandingreservoir cross-section height as linear (e.g., constant expansionangle, β), the cross-section height may expand non-linearly. Forexample, the reservoir may be defined at least partially by a dome-like(e.g., hemispherical) shape having variable expansion angles. Thecross-section height may expand in any shape.

The channel networks, e.g., as described above or elsewhere herein, canbe fluidly coupled to appropriate fluidic components. For example, theinlet channel segments are fluidly coupled to appropriate sources of thematerials they are to deliver to a channel junction. These sources mayinclude any of a variety of different fluidic components, from simplereservoirs defined in or connected to a body structure of a microfluidicdevice, to fluid conduits that deliver fluids from off-device sources,manifolds, fluid flow units (e.g., actuators, pumps, compressors) or thelike. Likewise, the outlet channel segment (e.g., channel segment 208,reservoir 604, etc.) may be fluidly coupled to a receiving vessel orconduit for the partitioned cells for subsequent processing. Again, thismay be a reservoir defined in the body of a microfluidic device, or itmay be a fluidic conduit for delivering the partitioned cells to asubsequent process operation, instrument or component.

The methods and systems described herein may be used to greatly increasethe efficiency of single cell applications and/or other applicationsreceiving droplet-based input. For example, following the sorting ofoccupied cells and/or appropriately-sized cells, subsequent operationsthat can be performed can include generation of amplification products,purification (e.g., via solid phase reversible immobilization (SPRI)),further processing (e.g., shearing, ligation of functional sequences,and subsequent amplification (e.g., via PCR)). These operations mayoccur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled for additional operations. Additionalreagents that may be co-partitioned along with the barcode bearing beadmay include oligonucleotides to block ribosomal RNA (rRNA) and nucleasesto digest genomic DNA from cells. Alternatively, rRNA removal agents maybe applied during additional processing operations. The configuration ofthe constructs generated by such a method can help minimize (or avoid)sequencing of the poly-T sequence during sequencing and/or sequence the5′ end of a polynucleotide sequence. The amplification products, forexample, first amplification products and/or second amplificationproducts, may be subject to sequencing for sequence analysis. In somecases, amplification may be performed using the Partial HairpinAmplification for Sequencing (PHASE) method.

A variety of applications require the evaluation of the presence andquantification of different analyte carrier or organism types within apopulation of analyte carriers, including, for example, microbiomeanalysis and characterization, environmental testing, food safetytesting, epidemiological analysis, e.g., in tracing contamination or thelike.

Computer Systems

The present disclosure provides computer systems that are programmed toimplement methods of the disclosure. FIG. 17 shows a computer system1701 that is programmed or otherwise configured to (i) control amicrofluidics system (e.g., fluid flow), (ii) sort occupied dropletsfrom unoccupied droplets, (iii) polymerize droplets, (iv) performsequencing applications, (v) generate and maintain a library of barcodedanalytes, and (vi) analyze results, such as to determine doublet rate orUMI purity. The computer system 1701 can be an electronic device of auser or a computer system that is remotely located with respect to theelectronic device. The electronic device can be a mobile electronicdevice.

The computer system 1701 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1705, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1701 also includes memory or memorylocation 1710 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1715 (e.g., hard disk), communicationinterface 1720 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1725, such as cache, othermemory, data storage and/or electronic display adapters. The memory1710, storage unit 1715, interface 1720 and peripheral devices 1725 arein communication with the CPU 1705 through a communication bus (solidlines), such as a motherboard. The storage unit 1715 can be a datastorage unit (or data repository) for storing data. The computer system1701 can be operatively coupled to a computer network (“network”) 1730with the aid of the communication interface 1720. The network 1730 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1730 insome cases is a telecommunication and/or data network. The network 1730can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1730, in some cases withthe aid of the computer system 1701, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1701 tobehave as a client or a server.

The CPU 1705 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1710. The instructionscan be directed to the CPU 1705, which can subsequently program orotherwise configure the CPU 1705 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1705 can includefetch, decode, execute, and writeback.

The CPU 1705 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1701 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1715 can store files, such as drivers, libraries andsaved programs. The storage unit 1715 can store user data, e.g., userpreferences and user programs. The computer system 1701 in some casescan include one or more additional data storage units that are externalto the computer system 1701, such as located on a remote server that isin communication with the computer system 1701 through an intranet orthe Internet.

The computer system 1701 can communicate with one or more remotecomputer systems through the network 1730. For instance, the computersystem 1701 can communicate with a remote computer system of a user(e.g., operator). Examples of remote computer systems include personalcomputers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad,Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone,Android-enabled device, Blackberry®), or personal digital assistants.The user can access the computer system 1701 via the network 1730.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1701, such as, for example, on thememory 1710 or electronic storage unit 1715. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1705. In some cases, thecode can be retrieved from the storage unit 1715 and stored on thememory 1710 for ready access by the processor 1705. In some situations,the electronic storage unit 1715 can be precluded, andmachine-executable instructions are stored on memory 1710.

The code can be pre-compiled and configured for use with a machinehaving a processor adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1701, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1701 can include or be in communication with anelectronic display 1735 that comprises a user interface (UI) 1740 forproviding, for example, results of sequencing analysis, such as thedoublet rate or UMI purity rate, or a control panel for one or moresingle cell application units or microfluidics units. Examples of UIsinclude, without limitation, a graphical user interface (GUI) andweb-based user interface.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1705. Thealgorithm can, for example, generate sequencing results and processsequencing results to determine, for example doublet rate or UMi purityrate, or other parameters of a single cell application.

Devices, systems, compositions and methods of the present disclosure maybe used for various applications, such as, for example, processing asingle analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g.,DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein)form a single cell. For example, an analyte carrier (e.g., a cell orcell bead) is partitioned in a partition (e.g., droplet), and multipleanalytes from the analyte carrier are processed for subsequentprocessing. The multiple analytes may be from the single cell. This mayenable, for example, simultaneous proteomic, transcriptomic and genomicanalysis of the cell.

EXAMPLES

The following examples merely illustrate the disclosure, and are notintended to limit the disclosure in any way.

Example 1 Production of Ligated RNA Constructs

This example demonstrates the production and analysis of ligated RNAconstructs that may be used to functionalize beads (e.g., gel beads thatcan be used as control beads).

RNA transcripts were first created from gblocks synthesized byIntegrated DNA Technologies (IDT), the sequences were designed with a T7primer on the 5′ end (e.g., TAATACGACTCACTATAGGG (SEQ ID NO: 3)) to beused later for RNA synthesis via in vitro transcription. The sequencesfor the gblocks used in this experiment can be seen below but anysequence may be used. The following two, exemplary RNA transcripts wereused:

(SEQ ID NO: 1, SynthCell #1)TAATACGACTCACTATAGGGAGCGTCTGTTATGATTCGGTTGTACCCGGAACAACTCCGTGCTCAGCTCAATGAAGGGCTGCGTGCAGCATATCTTTTACTTGGTAACGATCCTCTGTTATTGCAGGAAAGCCAGGACGCTGTTCGTCAGGTAGCTGCGGCACAAGGATTCGAAGAACACCACACTTTTTCCATTGATCCCAACACTGACTGGAATGCGATCTTTTCGTTATGCCAGGCTATGAGTCTGTTTGCCAGTCGACAAACGCTATTGCTGTTGTTACCAGAAAACGGACCGAATGCGGCGATCAATGAGCAACTTCTCACACTCACCGGACTTCTGCATGACGACCTGCTGTTGATCGTCCGCGGTAATAAATTAAGCAAAGCGCAAGAAAATGCCGCCTGGTTTACTGCGCTTGCGAATCGCAGCGTGCAGGTGACCTGTCAGACACCGGAGCAGGCTCAGCTTCCCCGCTGGGTTGCTGCGTAG, and(SEQ ID NO: 2, SynthCell #2)TAATACGACTCACTATAGGGAGCGTCTCTTATCATTCGGTTGTACCCGGAACAACTCCGATCGCAGCTCAATGAAGGGCTGCGCGATGCGTATCTTTTACTTGGTAACGATCCTCTGTTATTGCAG.

FIG. 11 shows how RNA was subsequently created from these transcripts instep 1101 using New England Biolabs HiScribe T7 ARCA mRNA Kit withtailing, however, any similar kit or kits may be used to complete invitro transcription, capping and A-tailing. This process created an RNAsequence starting at the last guanine of the T7 primer and ending with100-150 adenines on the 3′ end of the RNA. The RNA was cleaned andconcentrated using the Zymo Clean and Concentrator kit but any kit maybe used to clean the RNA. The concentration of synthesized RNA was thenquantified using a Nanodrop analyzer.

Ligation of the RNA molecule to an azide group was completed using T4DNA Ligase, a single stranded splint sequence containing 10 thymines anda 10 bp recognition sequence, and an oligo containing the complement 10bp recognition sequence on the 5′ end and an azide group on the 3′ endas shown in step 1102 of FIG. 11. The DNA ligase may be from anymanufacturer but the one used in the experiment came from Enzymatics andthe stock was 500 mg/mL. Additionally, the oligos may be synthesized byany manufacturer but these were made by IDT.

The ligation reaction contained 10 uM synthesized RNA, 10 uM splint, 40uM azide oligo, and 100 U/uL or 50 ug/uL ligase, Ligation Buffer wasthen added up to 250 uL. The reaction was incubated at 16 degrees for 1hour before 3 uL of EDTA was added to stop the reaction. The reactionwas then cleaned to remove small un-ligated oligos using the Zymo Cleanand Concentrator Kit. The concentration of ligated RNA was thenquantified using a Nanodrop analyzer.

This ligation was validated through digital droplet PCR (ddPCR). Primerand probes were designed to amplify the full length ligated product andcause fluorescence. FIG. 12A (resulted from the first RNA transcriptused) and FIG. 12B (resulted from the first RNA transcript used) showthe results of a ddPCR testing different ratios of components in theligation reaction with the first number representing the synthesizedRNA, the second representing the splint and the third representing theazide oligo. The number of dots above the pink line denotes the numberof full length ligated RNAs in the sample. Of the reaction ratiostested, the ratio of 1:1:4 appeared to have the highest number ofpositive events. The ligated RNA product was stored at −80 until usedfor clicking onto the gel beads (e.g., control beads).

This example demonstrates that nucleic acid molecules (e.g., RNAmolecules) may be efficiently functionalized with various moieties, suchas those enabling functionalization of beads such as control beads.

Example 2 Production of RNA-functionalized Gel Beads

This example demonstrates the production and analysis ofRNA-functionalized gel beads (e.g., those that can be used as controlbeads) using azide-modified RNA samples, e.g., those that were producedas described above in EXAMPLE 1.

First, 30 μm RNA-functionalized gel beads were produced using methodsknown in the art (see, e.g., U.S. Pat. Nos. 9,695,468, 9,951,386, and9,689,024 and U.S. Patent Application Publication Nos. 2015/0376609,2017/0321252, 2018/0016634, 2018/0216162, and 2018/0371540, which areincorporated herein by reference in their entirety) with the exceptionthat the primer on the gel bead was replaced with a primer containing analkyne group which could be used later in the click chemistry reaction.Additionally, Bis-acrylamide was used in place of Bis-Cys to keep thebeads from dissolving in DTT. The aqueous solution was formulated sothat the final gel beads had an alkyne concentration of 20 uM.

The functionalized gel beads were also run through the flow cam toverify the size, these functionalized gel beads did not swell as much asunmodified genome gel beads, the average sizes over three runs measuring25,000-30,000 gel beads per run was 28.24 um. The smaller size is likelydue to the replacement of bis-cys for bis-acrylamide.

Click chemistry was used to attach the azide-functionalized RNAconstructs onto the beads and was completed using stock solutions of theligand THPTA at 50 mM, 10 mM copper acetate, 100 mM sodium ascorbate,0.16 mM ligated RNA, and the 20 uM gel beads (see e.g., step 1301 shownin FIG. 13). The final reaction was 300 uL 1.25 mM THPTA, 0.25 mMCuAcO₄, 5.0 mM sodium ascorbate and 0.04 mM ligated RNA, and 20 uM gelbeads. This reaction was incubated at room temperature while rolling at30 rpm for 2 hours, then 4 uL of EDTA were added and the gel beads werewashed in buffer.

Validation of the click chemistry reaction and thus validation ofattachment of the RNA molecules to the beads was completed through probehybridization of the Poly-A tail using a Poly-T oligo with an Alexa647probe synthesized by IDT. 50 uL of the washed and packed gel beads weremixed with 50 uL of 12 uM polyT probe. The reaction was placed on athermomixer at 95° C. and covered with a lid to protect the probe fromlight. The thermomixer was then set to 25° C. and 1000 rpm and thereaction was slowly cooled while shaking. After cooling the mixture wascleaned again with buffer to remove unclicked RNA and probe. 5 uL of themixture were added to 1495 uL buffer in a Guava analysis tube which weremixed by vortexing then placed in the Guava machine. Three technicalreplicates were tested. If the ligation and click chemistry weresuccessful there should be a polyA sequence attached to the gel beadwhich would then hybridize to the PolyT probe producing increasedfluorescence visible on the Guava.

FIGS. 14A-D show the results of this test on the Guava, the location ofthe peak on the X-axis denotes level of red fluorescence. The negativecontrol (FIG. 14A) went through the ligation and clicking processes butdid not include an azide so no clicking would be possible but there wasa PolyA sequence in the mix. The observed right shift in thefluorescence signal (FIG. 14A-C) which indicated an increase influorescence confirmed that the gel beads did contain the synthesizedRNA and thus indicated attachment of nucleic acid molecules to thebeads.

This example demonstrates that the azide-functionalized RNA moleculesproduced in EXAMPLE 1 can be efficiently attached (e.g., chemicallylinked via click chemistry and/or covalent or non-covalent bondformation) to gel beads. Such functionalized gel beads may be used ascontrol beads, for example, as control elements in single-cell analysesprocesses.

Example 3 Use of RNA-Functionalized Gel Beads in Single-Cell Sequencing

This example demonstrates the use of synthetic cells as produced abovein EXAMPLE 2 comprising RNA molecule(s) (e.g., attached to theirsurface) in a single-cell sequencing experiment to determine whether thetranscripts can be detected after sequencing.

The synthetic cells used in the experiment were generated using thetranscript sequences described in EXAMPLE 1. The ratio of thetranscripts with SEQ ID NO: 1 and SEQ ID NO: 2 on a bead was about 1:2.

For this study, approximately 1000 synthetic cells were used. cDNA wasproduced and the size of the peaks matched the expected transcriptsizes, with an additional peak at 75 bp which is likely primer dimer.The bioanalyzer traces are shown in FIG. 15.

Libraries were then made from the cDNA and the corresponding bioanalyzertrace in FIG. 16 shows the results. The cDNA sequences were randomlyfragmented, possibly explaining the peak width observed in this study.The first two peaks in the test samples and the first negative controlare likely from the primer dimer seen in the cDNA, while the second twopeaks, corresponding to 311 bp and 674 bp, respectively, were seen onlyin the test samples, and thus correspond with a high likelihood to theexpected transcripts.

Subsequently, both test sample's libraries were submitted for sequencingon the MiSeq analyzer. Both samples produced high numbers of R2 sequencebut 3% of the reads mapped to a part of one or both transcripts. Thetranscript sequences (see EXAMPLE 1) may have been too similar in theirsequence to determine the ratio between the two sequences, however, theexpected transcripts were clearly observed in the sequencing analysis.

Thus, the data presented herein demonstrate that the synthetic cellsdescribed herein can be used as a library preparation tool and/or assequencing standards in single-cell analyses biological samples. Thedata further show that a nucleic acid sequence that is attached to a gelbead or control bead via click chemistry can be recovered duringsequencing as if the nucleic acid sequence belonged to a cell (e.g., acell of a biological sample to be analyzed in a single-cell analysis).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

What is claimed is:
 1. A method for analyzing a single cell process in acell sample, comprising: (a) using said single cell process to partition(i) a mixed sample comprising a plurality of cells, a plurality of firstcontrol beads, and a plurality of second control beads and (ii) aplurality of barcode beads, thereby providing a plurality of partitions,wherein said plurality of partitions comprises a first partition and asecond partition, wherein said plurality of first control beadscomprises a plurality of first nucleic acid molecules comprising firstknown control sequences, wherein said plurality of second control beadscomprises a plurality of second nucleic acid molecules comprising secondknown control sequences, wherein a first subset of said first knowncontrol sequences and a second subset of said second known controlsequences are mutually exclusive, wherein a ratio between a firstconcentration of said plurality of first control beads and a secondconcentration of said plurality of said second control beads in saidmixed sample is a known ratio; (b) generating a plurality of barcodednucleic acid molecules in said plurality of partitions, wherein (i) insaid first partition, a plurality of barcoded first control nucleic acidmolecules is generated using (A) first nucleic acid molecules of a firstcontrol bead of said plurality of first control beads, wherein saidplurality of first nucleic acid molecules comprises said first nucleicacid molecules, and (B) first nucleic acid barcode molecules, whichcomprise first barcode sequences, of a first barcode bead of saidplurality of barcode beads, and (ii) in said second partition, aplurality of barcoded second control nucleic acid molecules is generatedusing (A) second nucleic acid molecules of a second control bead of saidplurality of second control beads, wherein said plurality of secondnucleic acid molecules comprises said second nucleic acid molecules, and(B) second nucleic acid barcode molecules, which comprise second barcodesequences, of a second barcode bead of said plurality of barcode beads(c) sequencing said plurality of barcoded nucleic acid molecules, orderivatives thereof, to generate sequencing results to identify (i) saidfirst known control sequences and said first barcode sequences from saidplurality of barcoded first control nucleic acid molecules and (ii) saidsecond known control sequences and said second barcode sequences fromsaid plurality of barcoded second control nucleic acid molecules; and(d) using said sequencing results and said known ratio to analyze anidentifier purity or a doublet rate of said single cell process, whereinsaid identifier purity is indicative of any change to a set ofidentifier sequences associated with said first known control sequencesor second known control sequences during said single cell process. 2.The method of claim 1, wherein (d) comprises comparing a frequency ofsaid first known control sequences and a frequency of said second knowncontrol sequences to analyze said single cell process.
 3. The method ofclaim 1, wherein (d) comprises determining said doublet rate and saididentifier purity of said single cell process.
 4. The method of claim 1,wherein said first control bead has a size within 25% deviation from anaverage size of said plurality of cells.
 5. The method of claim 1,wherein said first known control sequences are derived from a firstmammalian species and said second known control sequences are derivedfrom a second mammalian species.
 6. The method of claim 1, furthercomprising, prior to (a), mixing cells from said plurality of cells withcontrol beads of said plurality of first control beads and control beadsof said plurality of second control beads.
 7. The method of claim 1,wherein a ratio of a concentration of said cells to said firstconcentration in said mixed sample is known.
 8. The method of claim 1,wherein (i) said first known control sequences vary in length,concentration, and/or GC content among each other in said first knowncontrol sequences, and wherein (ii) said second known control sequencesvary in length, concentration, and/or GC content among each other insaid second known control sequences.
 9. The method of claim 1, wherein agiven barcode bead of said plurality of barcode beads comprises aplurality of nucleic acid barcode molecules, wherein each of saidplurality of nucleic acid barcode molecules comprises an identifiersequence associated with said each of said plurality of nucleic acidbarcode molecules.
 10. The method of claim 1, further comprisingidentifying a first set of identifiers associated with said first knowncontrol sequences and a second set of identifiers associated with saidsecond known control sequences, and processing said first set ofidentifiers and said second set of identifiers to determine saididentifier purity for said single cell process.
 11. The method of claim1, wherein said first control bead has a size within 25% deviation froman average size of said plurality of cells.
 12. The method of claim 1,wherein said first control bead has a size between from about 15micrometers to about 60 micrometers.
 13. The method of claim 1, whereina given barcode bead from said plurality of barcode beads comprises aplurality of nucleic acid barcode molecules each comprising a commonbarcode sequence, wherein said common barcode sequence is different fromcommon barcode sequences of other barcode beads of said plurality ofbarcode beads.
 14. The method of claim 1, wherein said first barcodebead comprises said first nucleic acid barcode molecules releasablyattached thereto, and wherein said first nucleic acid barcode moleculesare released from said first barcode bead.
 15. The method of claim 1,wherein said plurality of barcode beads is a plurality of gel beads. 16.The method of claim 1, wherein said first control bead is a gel bead.17. The method of claim 1, wherein said first control bead comprises afirst functional group, and said first nucleic acid molecules comprise asecond functional group, and wherein said first nucleic acid moleculesare attached to said first control bead by reacting said firstfunctional group with said second functional group.
 18. The method ofclaim 17, wherein (i) said first functional group is an alkyne, atrans-cyclooctene, an avidin, or any combination thereof, and (ii) saidsecond functional group is an azide, a tetrazine, a biotin, or anycombination thereof.
 19. The method of claim 17, wherein said firstfunctional group reacts with said second functional group in a clickreaction, wherein said click reaction is a copper-catalyzed azide-alkynecycloaddition reaction, an inverse-electron demand Diels-Alder reaction,an avidin-biotin interaction, or a copper-catalyzed azide-alkynecycloaddition reaction.
 20. The method of claim 1, wherein said firstnucleic acid molecules is within said first control bead.
 21. The methodof claim 1, wherein said first known control sequences are synthetic.22. The method of claim 1, wherein said first known control sequencesare derived from a biological sample.
 23. The method of claim 1, whereinsaid first control bead comprises a plurality of protein-DNA complexesand said first nucleic acid molecules are parts of said plurality ofprotein-DNA complexes, wherein said first known control sequencescomprise defined protein binding sites.
 24. The method of claim 1,wherein said plurality of cells is or comprises a plurality of cellbeads.
 25. The method of claim 1, wherein said plurality of partitionsis a plurality of droplets and/or a plurality of wells.
 26. The methodof claim 1, wherein said plurality of cells comprises a messengerribonucleic acid (mRNA) molecule, wherein (b) comprises generating abarcoded mRNA molecule, or derivative thereof, using said mRNA moleculeand a third nucleic acid barcode molecule, which comprises a thirdbarcode sequence, of a third barcode bead of said plurality of barcodebeads, and wherein (c) comprises sequencing said barcoded mRNA molecule,or derivative thereof, to identify a sequence of said mRNA molecule andsaid third barcode sequence.
 27. The method of claim 1, wherein (d)comprises determining said identifier purity of said single cellprocess, and further comprising determining a polymerase chain reaction(PCR) chimerism rate of said single cell process using said identifierpurity.
 28. The method of claim 1, wherein said plurality of partitionscomprises a third partition, wherein in (b), in said third partition, aplurality of barcoded nucleic acid molecules is generated using templatenucleic acid molecules of a cell of said plurality of cells and thirdnucleic acid barcode molecules, which comprise third barcode sequences,of a third barcode bead of said plurality of barcode beads.